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1. Carnegie Mellon IGEM Why traditional cloning We chose this method because of the hundreds available enzymes each with a specific target sequence which gives us a predictable resulting end In addition it is relatively cheap therefore it is very efficient to use this method given our budget oTa E RS s f G I A 18 Materials Restriction Enzymes NEB Reaction Buffer often supplied with enzymes gBlock IDT PCR product annealed oligos IDT digested DNA Plasmid backbone ADDgene IDT NEB o Common plasmid backbones are part of the pUC and pET series which are easy to use for beginners To assist with cloning in terms of finding restriction sites we often use software o A plasmid editor ApE is used to sequence the DNA and is easy to use o Genome complier gives access to iGEM teams and advanced features but it is hard to understand for beginner applicants Experiences How did you experience working with this cloning method It worked really well We did not have much trouble when going through our method but there were some struggles especially when it came to obtaining the fluorescence wanted from the plasmids used We had a difficult time transforming the correct plasmid into the chassis but it was pretty simple isolating the correct plasmid What was the most difficult task The mos
2. o Q Advantages Disadvantages Gel purification of the digested parts is e Ifthe parts do not already come in the not necessary for good results correct vectors cloning them into the Saves time when up scaling proper backbone eliminates much of the When done correctly 9796 of colonies speed and convenience of 3A Assembly will have the desired assembly 2 Yields a lower concentration of DNA Works even with small pieces of DNA than standard assembly because three It s very iGEM friendly since iGEM pieces are ligated rather than two already provides the required linearized backbones Further applications e Changing the resistance marker This method can be useful if you wish to transfer parts into backbones with alternative antibiotic resistance for example if you wish to move parts from backbones providing am picillin resistance to those providing chloramphenicol resistance Assembling DNA from PCR A modified version of 3A assembly can also be used if amplifying DNA in two halves using PCR After purification you can treat the two amplified DNA fragments as digestions from BioBricks if you include the BioBrick prefix and suffix in the primers Then you can assem ble them directly into a BioBrick plasmid backbone using the same protocol as 3A assembly Frequently Asked Questions e What does 3A stand for It stands for 3 Antibiotic after the 3 antibiotic markers found in the backbones of your construct Is it n
3. E Ligase o C TCGAG GAGCT C a Figure 1 Traditional cloning work flow restriction digestions of vector backbone purple and gene of interest pink yields fragments with sticky ends These fragments can be ligated using T4 ligase Workflow Traditional cloning See Figure 1 isa method to clone a gene of interest into a vector of choice For a successful cloning both the vector and the insert have to be digested with compatible re striction enzymes to create complementary overhangs By using restriction enzymes that cre ate different overhangs the gene of interest can be cloned into the vector in a specific direction The resulting complementary sticky ends can anneal and ligate by T4 ligase to create a circular vector containing the gene of interest which can be transformed into competent cells To increase cloning efficiency the vector backbone typically gets dephosphorylated preventing self ligation of the vector as the 5 phosphate group is catalytically required by the T4 ligase The Cutters Traditional cloning is based on restriction endonucleases enzymes that cut DNA at specific recognition sites 1 Usually type II restriction enzymes Figure 2 are used which cut the DNA inside a specific palindromic recognition sequence The way restriction enzymes cut DNA can differ in the following ways 2 e Blunt end cutters Enzymes that cut DNA creating no overhangs so called blunt ends
4. 5 Clontech provides an online tool that simplifies In Fusion PCR primer design for standard cloning reactions Simply provide your vector sequence the restriction enzyme s used to linearize the vector if that is the chosen method for linearization and the primer sequence required to amplify your region of interest Following PCR verify by agarose gel electrophoresis that your target fragment has been am plified If a single band of the desired size is obtained you can EITHER spin column purify OR treat your PCR product with In Fusion Cloning Enhancer However if non specific back ground or multiple bands are visible on your gel isolate your target fragment by gel extraction then spin column purify The setup of the In Fusion cloning reaction Mix and the transformation that follows depend on the chosen method Refer to the In Fusion HD Cloning Kit guide Materials For the design of our experiments we used the following materials e In Fusion HD Cloning Kits 2 ul of 5X In Fusion HD Enzyme Premix X ul of Linearized Vector X ul of insert X ul of dH O to a total reaction volume of 10 uL e Primer design software we used Serial Cloner e Miniprep Kit We used the OIAprep Spin Miniprep Kit Experiences e How did you experience working with this cloning method Everything went as expected What was the most difficult task The main difficulty was to design PCR primers that fitted with the help of some
5. Figure 45 Team photo of the Stockholm iGEM team 2015 From back to front Hugi Asgeirsson Maxi milian Karlander Pontus H jer Karol Kugiejko Radoslaw Gora Sarah Wideman Carmen Gallo lvarez Manon Ricard Linnea Osterberg Alison Shea Baxley Utsa Karmakar Felix C Richter Not in the picture Denise Strand Naz Karadag Katrine Horne Iversen Mona Hassan Hugo Morales Tello Axel Bergenstrale
6. From left to right Lizzie Mark Gaia Sandi Harrison and Mahiar in the middle Yeast Recombination Plasmid linearization nnsisms 2s Nucleus Transformation gt aa n into yeast BN Ex Vacuole Recombination L m mu OO 64 Yeast Recombination Figure 35 The first step is to linearize the plasmid either through restriction digestion or PCR ampli fication After adding some chemicals to make the yeast competent add all of the vector and inserts with the weakened yeast and transform the yeast The inserts and vector will recombine into a plasmid through homologous recombination PCR screen of the junctions can be used to analyze if the transfor mation worked Introduction Homologous recombination is the exchange of DNA strands of similar or identical nucleotide sequences This genetic recombination was first hinted at in the 1900 s when William Bateson and Reginald Punnett noticed that certain traits tended to be inherited together and then later in 1911 Thomas Morgan noticed that some of these genetically linked traits can be on occasion be inherited separately This led Morgan to hypothesize that there are crossovers between chromosomes This was proved to be correct by Barbara McClintock and Harriet Creighton in the 1930 s when they demonstrated the crossover during meiosis As the years progressed it was shown that all three domains of life as well as viruses have
7. Use a maximum total volume of 15 uL Use equimolar amounts of all inserts and vector higher ratio may result in mis matched ligation Make sure that all DNA samples are pure and free of contaminants miniprep and DNA extraction kits are adequate compared to miniprep Mix sample by pipetting up and down or gentle tapping Use 5 8 uL for transformation Overall we believe that Golden Gate Assembly is a promising method and while it is at its early stages of optimisation and development it can be a very convenient and effective way of constructing clones with large number of inserts We hope that by sharing this information we can make available more information for further optimisation and devel opment of this method Protocols iGEM Sydney has published protocols for Golden Gate Assembly on their iGEM Wiki These protocols include e Golden Gate Assembly Protocol e Golden Gate Assembly design considerations The iGEM Sydney team This year the Sydney iGEM team is working with the ethene monooxygenase enzyme that performs the epoxide reaction converting ethylene to ethylene oxide This enzyme is only natively found in Mycobacterium smegmatis however this host is difficult to work with on an industrial scale Our main goal is to optimise expression of this enzyme in Escherichia coli More information regarding our team and project can be found in our website http 2015 igem org Team Sydney Australia Figure 34 Photo of iGEM Sydney 2015
8. culture tests RbCI method and cloning Each of the protocols is described in more detail on the wiki of team Toulouse Iterative Capped Assembly Introduction Iterative Capped Assembly ICA is a cloning method that is used to sequentially assemble long repetitive DNA sequences This technique was developed by Briggs et al in 2012 as a method to assemble Transcription Activator Like Effector Nucleases TALENSs which are sequence specific DNA binding proteins that consist of multiple repetitive monomers 1 Each repeat monomer is responsible for binding to a specific nucleotide in the target sequence Due to the repetitive nature of TALE genes conventional PCR is unable to reliably amplify these sequenc es due to non specific primer binding Although ICA was developed using TALE construction as a model problem this technique can be used to construct long repetitive DNA constructs in a directly controllable fashion ICA assembles repetitive sequences one monomer at a time while preventing the elongation of incomplete nucleotide chains The full length sequence is flanked by unique primer annealing sites which allows the PCR amplification of the final product This entire process is performed using a solid substrate which greatly facilitates the construction of long sequences 44 Iterative Capped Assembly ur ur ali pii UU Final step 1 Repetition of i l i Steps 3 J Sa mS ar ar ee Figure 23 Workflow overview of
9. i o mE A d n NeARm SB S Ligation 4 Figure 36 The first step in homologous recombination HR is for the modified inserts to align with homologous regions A double stranded break will be induced After some processing of the vector one of the strands of the insert will invade the space left from the induced break to provide a template to bridge the gap SOR ig cic ap Highly efficient seamless method Several days to finish procedure No purification steps Difficult to add lengthy tags Only need enzymes and plates no kits Need eukaryote vector or yeast origin of necessary replication Fragment design similar to Gibson As Efficiency decreases as the number of sembly if yeast recombination does not fragments increase work Difficult to interchange fragments in yeast Need a shaker and incubator at 30 C Frequently Asked Questions e Does the size of the insert affect recombination efficiency Yes increased size of insert will decrease the efficiency because it will increase the plasmid size which yields lower transformation efficiency Our mentor Dr Sarah Perdue was able to transform an 18 kb plasmid into yeast and still had a decent transformation efficiency e How many fragments can be assembled The biggest achievement was 25 fragments ranging in size from 17 kb to 35 kb 7 however we would not recommend trying this as your efficiency tends to decrease as you increase the number of fragments Tha
10. ri iGEM team h L k L ail il p s E 4 4 h E 3 Fisure 18 Team photo of York from left to right back row Ivan Gyulev Jun Hee Jung Adam Brain Joseph Tresise Matthew Higgins front row Katy Davis Erin Cullen Kristina Aare Mat Milner Abi Robowtham Clare Draper Liz Alexianu Introduction In Fusion Cloning In Fusion cloning method is used for directional cloning of one or more fragments of DNA into any vector The cornerstone of In Fusion cloning technology is Clontech s pro prietary In Fusion Enzyme which fuses DNA fragments by recognizing a 15 bp overlap at their ends Figure 19 This 15 bp overlap can be engineered by designing primers for amplification of the desired sequences Step 2 Design PCR primers for your gene of interest with 15 bp extensions 5 that are complementary to the ends of the linearized vector Step 1 Select a base vector and identify the insertion site Linearize the vector by restriction enzyme digestion or inverse PCR and purify Step 3 Amplify your gene of 15bp 15bp interest Verify on agarose gel _ anm o0 S that your target DNA has been EN B EE far amplified and determine the E as integrity of the PCR product z PCR or O Step 4 Spin column purify d your PCR product OR treat it EN Ts with Cloning Enhancer z Q Step 5 Set up your In Fusion cloning reaction 38 Step 6 Transform compete
11. DNA concentration since yeast grow at a much slower rate than yeast We doubled our transformations Ci O D e c qe Ee O Q QD a n V gt NO NO Useful additional information amp resources Minessota s mentor Dr Sarah Perdue made her own protocol for yeast recombination by borrowing from the following OpenWetWare protocol The protocol also has some tips at the bottom Transformation which formed a source of inspiration for Minessota s proto cols This is a step by step protocol for the yeast transformation Addgene known as the nonprofit plasmid repository has posted an article on Yeast Vectors This information can be used if you want to make a yeast vector from any other plasmid by inserting a yeast origin of replication IGEM Minnesota Why Yeast Recombination Team Minnesota 2015 choose this cloning technique because we need to express our genes in a eukaryotic vector and this is the preferred technique of one of our mentors While this process has several more steps than other methods such as direct ligation or Gibson Assembly it tends to have a higher rate of success than other methods Thanks to the gBlocks from IDT it makes this process very easy to use as you can decide how long the homologous sequences are going to be and where the fragments should go This process has been fairly easy to trou bleshoot because yeast uptake DNA and homologous recombine so readily Experiences e How did
12. For our sensor we sketch a few applica tion scenarios It can for example be used to diagnose Q fever used within the intestinal tract and used as a excretor of pesticides qp am b 4 poli y Yih T y 2 Pn TA 4 i E rb E x z t t E j ri Figure 15 Team photo of iGEM TU Eindhoven in front of the Institute for Complex Molecular Scienc es ICMS Top row from left to right Femke Vaassen Laura van Smeden Sjoerd Nooijens Hans de Ferrante Cas van der Putten and Jan Willem Muller Bottom row from left to right Kwankwan Zhu Yeshi de Bruin Elles Elschot Laura Jacobs and Esther van Leeuwen Protocols e Vector Linearization Protocol which can be followed to obtain a linear vector This protocol consists of a PCR step an optional CALL Dpnl digestion step an optional PCR purification step a Nano H r5 Te Drop step and an optional gel electrophoresis step TT conl e NEBuilder HiFi Assembly Protocol which makes use of New T am England Biolabs HiFi kit in order to assemble DNA The NEBuild M pAERC er kit can be seen as superior to the original Gibson Assembly kit WILLE ar as it contains a high fidelity polymerase IGEM Paris Pasteur Why Gibson Assembly From the beginning we realized that the most difficult part of our project would be to assem ble all the needed genes as well as the proper intersequences containi
13. J Schatz D Botstein Plasmid construction by homologous recombination in yeast Gene 58 201 16 1987 4 C K Raymond T A Pownder S L Sexson General method for plasmid construction using homolo gous recombination Biotechniques 26 134 8 140 1 1999 5 D L Marykwas S E Passmore Mapping by multifragment cloning in vivo Proc Natl Acad Sci U S A 92 11701 5 1995 6 T Ebersole et al Rapid generation of long synthetic tandem repeats and its application for analysis in human artificial chromosome formation Nucleic Acids Res 33 e130 2005 7 D G Gibson et al One step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome Proc Natl Acad Sci U S A 105 20404 9 2008 8 S B Hua M Qiu E Chan L Zhu Y Luo Minimum length of sequence homology required for in vivo cloning by homologous recombination in yeast Plasmid 38 91 6 1997 LA Cloning Reaction buffer TOPO Vector 5 minute reaction m gu ES Figure 38 Overview of general TOPO cloning Combine your PCR product with the applicable TOPO cloning vector in the provided reaction buffer and incubate for 5 minutes Topoisomerase I purple diamond relegates the PCR product to the TOPO vector while detaching itself during the process Now that your cloning vector is ready you can transform it into your competent cells for further applica tions
14. NEBuilder kits This manual features among others NT Asked Questions Pro OFF a a 40 When you have a dip because your Gibson Assembly failed it is very easy IH E TA Fr to get enthusiastic again with the Gibson Assembly song by the Cambridge EF MP elis uc iGEM Team of 2010 Se jmd eu yn Q Qu c A FF md le 6C IGEM TU Emdhoven Why Gibson Assembly In previous years iGEM TU Eindhoven teams have always used traditional cloning and Bi oBricking as their assembly standards for cloning These methods are also frequently used within the labs of our university We were ready to take on the challenge of introducing our university to a fairly new cloning technique Moreover Integrated DNA Technologies and New England Biolabs offered to sponsor teams with gBlocks and the NEBuilder Cloning Kit enabling us to try dive into Gibson Assembly It must be noted however that we were not prepared to fully bet on Gibson Assembly both due to the fact that we had never used it before and that it is hard to troubleshoot As a back up plan we devised to use traditional cloning and for the construction of the vectors which we sent out to iGEM HQ we used BioBricking Materials Q For the design of our experiments we used the following materials d e ThepETDuet 1 vector designed by Novagen e ThegBlocksQ offered by Integrated DNA Technologies e The NEBuilder Cloning Kit supplied by New En
15. a handful of changes The protocol states to digest the parts for 30 minutes but our team typically increased this time to 1 2 hours before proceeding to heat killing or even up to 4 hours if the schedule permitted We also used a larger amount of restriction enzymes typically 0 8 1 0 uL of each enzyme per tube These changes were made to ensure that as much of our DNA as possible was properly digested Also Open WetWare notes that 3A Assembly can sometimes fail due to genomic DNA being cloned into the construction plasmid and advises phosphatase treatment of the linear plasmid construct as a remedy 5 Wad ONIN ec Protocols OpenWetWare has a page dedicated to 3A Assembly The page contains some explanatory graphs good protocols amp troubleshooting tricks and should be a good starting point for beginning iGEM teams iGEM Illinois has compiled the used protocols on their iGEM Wiki Take a look at their wiki to find an overview of these protocols 3A Assembly plays a central role within the iGEM competition as the iGEM foundation provides participating teams with BioBricks following the 3A Assembly standard To get teams started iGEM has published some protocols on their website 3A Assembly Experiences e Howdid you experience working with this cloning method Due to time restrictions we ended up using our cloning method only once not counting a practice run in our training sessions at the beginning
16. a lower efficiency Is it necessary to digest the template when using PCR to linearize the vector No digestion of the template is optional as long as a minimal amount of template vector is used think 0 5 1 0 ng of template The template will be transformed into competent cells If you choose to skip digestion of the template it is wise to analyze additional colonies using colony PCR to ensure that your DNA fragments have been correctly inserted What size should my overlaps have Thisreally depends on the cloning kit you are using It is best to read out the manual supplied with your cloning kit to find out what overlap size works best In general greater overlaps result in a greater transformation efficiency How reliable is Gibson Assembly Gibson Assembly is quite a robust and proven cloning technique However as the method is homology based some fragments are harder to assemble than other fragments In addition troubleshooting is quite difficult as the reaction incorporates multiple steps into a single tube reaction Hence many life science firms advise to have a back up plan should Gibson Assem bly fail Useful additional information amp resources Integrated DNA Technologies has published Gibson Assembly Cloning Protocols This document compiles protocols and instructions for the use of Gibson Assembly in combination with gBlocks Gene Fragments m aero SIN az New England Biolabs provide an instruction manual with its
17. called the genetic tape recorder the SCRIBE system uses genomic recombination events to measure the frequency intensity or duration of the inducer that the chassis organism encountered This represents an advantage over traditional biosensors which can generally say whether an inducer was encountered but cannot indicate how much or how long 48 amp 02 BE m T x N Umm lan Figure 10 Team Photo of UIUC iGEM From the left Jess Beaudoin advisor Linyang Andrew Ju Joshua Cheng Caroline Blassick Dr Yong Su Jin advisor Miranda Dawson Ashwin Pillai Noah Flynn Michelle Goettge advisor Erik Anderson advisor James Blondin Todd Freestone advisor Pierce Hadley Zach Costliow advisor Sameer Andani Aru Singh Gibson Assembly 26 Gibson Assembly Sa eee Vector linearization a es L EE LE Qa Picking amp Lysing Gibson Assembly amp the bacteria Direct transformation Amplifying the Verification of insert insert Colony PCR size Colony PCR D i o Figure 11 General workflow of Gibson Assembly The first step consists of linearizing the vector using either PCR or digestion by restriction enzymes Next the linearized vector and dsDNA fragments are introduced in a tube with the Gibson Assembly Master Mix and incubated at 50 C The resulting mix ture is transformed into competent ce
18. sticky ends differ for each different unit The Pieces A basic biobrick for ICA consists of a gene monomer flanked by Bsal recognition and cleavage sites While each of the core monomer is identical the restriction sites are oriented such that digestion with Bsal yields distinct sticky ends for each of the three types of units These pieces must be digested to reveal the sticky ends before assembly see Figure 25 Accesory pieces in ICA include the initiator streptavidin coated beads the terminator and the capping oligos see Figure 26 e Theinitiator a dsDNA fragment made by annealing two ssDNA oligos together The ini tiator is designed such that one end is biotinylated for conjugation to streptavidin coated beads The other has a sticky overhang and is designed to anneal to the forward sticky end of the A type monomer unit This end is 5 phosphorylated to enable ligation The initiator also contains a primer binding site that can be used for PCR amplification as well as other accesory sequences such as affinity tags and the BioBrick prefix Streptavidin coated beads these beads serve as a solid support for the elongating DNA chain during ICA The biotinylated end of the initiator binds to streptavidin to anchor the nascent construct The ability to physically seperate the DNA from solution is needed due to repeated wash and ligation steps used during ICA The terminator a dsDNA fragment that is constructed similarly to
19. the preferred method for cloning several inserts into a vector our team decided to use an alternative method due to mixed reports of success of Gibson Assembly and the fact that each of the inserts were pretty large Also we thought it would be exciting to use and test out a novel method Materials During our experiments we used the following NEB Bsal Bsal HF restriction enzyme 20 U uL NEB T4 DNA Ligase concentrated 2000 U uL NEB 10X T4 DNA Ligase reaction buffer 500 mM Tris HCl 100 mM MgCl2 10 mM ATP 100 mM DTT pH 7 5 at 25 C Insert equimolar amounts to vector Digested PCR linearised vector e g 50 ng Bsal HF was used in our experiments normal Bsal should work as well according to liter ature Design concideration The same design protocol as prescribed at the NEB website was followed T If there are 3 inserts design each end that needs to be put together to contain 4 bp over lapping overhangs 2 The left hand side overhang of the first insert and the right hand side overhang of the last insert need to overlap with the overhangs of the digested vector 3 Start by selecting the enzymes you would like to digest your vector e g Spel EcoRI and design their corresponding overhangs on the inserts to overlap with them 4 Make sure to place the Bsal recognition site outside of the ORF regions so that it is di gested and removed from the final DNA e g place it left hand side of the green overhang and right ha
20. this biological mechanism In 1978 Dr Hinnen demonstrated that yeast was able to recombine plasmids followed in 1981 by Dr Orr Weaver who reported the mechanistic studies of how yeast undergoes homologous recombination 1 2 This technique is preferred by eukaryotic cells to repair double stranded breaks as they are able to recover nucleotides that are lost due to the break if the other homol ogous strand remains less damaged Yeast in particular frequently and efficiently undergo homologous recombination which makes it a good tool to construct plasmids even from multi ple overlapping DNA fragments 3 6 Points of interests Requires linearized eukaryote vector and dsDNA fragments dsDNA need to have an overlapping region of at least 29 base pairs on both sides 40 base pairs is recommended Overlapping region can be added as part of gene synthesis gBlock or primers can be used to add the overlapping sequence through PCR if the desired product is already cloned Repetitive sequences greater than 15 base pairs anywhere in the vector or fragments should be avoided to prevent undesired recombination during cloning process or later More fragments decrease efficiency Yeast have a higher level of translation when the Kozak sequence ACC is added before the start codon Induced Double Strand Break o L M M Processing amp GolI Selection Marker Bee 4 5 em yn le oF _ _ _ j f_ zz
21. to optimize your protocol Many labs simply follow whatever protocol has been passed down over the years without searching for themselves if there are better ways these protocols can be done Investing the time to try other protocols make tweaks to each step and find out what works best can pay off in the long term IGEM Vanderbilt We are the iGEM team from Vanderbilt University in Nashville TN The group is made up of about 8 undergraduates majoring in sciences ranging from biology to math This year s pro ject is the modulation of evolutionary potential where we take DNA sequences and optimize them to reduce the possibility of mutation We then apply other principles of genetic stability to gene circuits and entire organisms To this end we have written an algorithm that incorpo rates decades of DNA damage research to generate the best possible sequence Our research has substantial implications in the field of biosafety commercial biomanufacturing and DNA therapeutics e i o Figure 6 Team photo of iGEM Vanderbilt Photograph Left to Right Daniel McClanahan Stephen Lee Ophir Ospovat Jarrod Shilts Sikandar Raza Protocols Protocols describing the various cloning steps performed by the iGEM team Vanderbilt These protocols include important digestion ligation and purification steps
22. with empty vector and the vector with ligated insert are transformed into suitable bacteria If colonies are only present in the vector with gene insert it means empty vector did not self ligate Experiences e How did you experience working with this cloning method It is quite laborious compared to the newest cloning strategies however traditional cloning provides a cheap and rather easy way of cloning What was the most difficult task Finding the right conditions and buffers to perform a double digest can sometimes be a little tricky although this is much simplified using the double digest finder by NEB Did the cloning method work as expected Yes usually everything worked with a high efficiency Still every cloning step was followed by control restriction digests to verify correct cloning and sort out clones with plasmids without insert What was the biggest achievement using this cloning method Successful cloning of insert into target vector What would be your tips and tricks if other teams are going to use this method Standard cloning is more or less foolproof as long as you stick to the protocol you can hardly do anything wrong We would recommend this method if you want a simple method to im plement your pathway constructs and extra time to focus on other experimental or outside the lab aspects of your project Ke m E o gt g D ua f m m D T e F QU N Figure 4 iGEM Manchester Graz s vector asse
23. C monomers is critical We used T7 DNA Ligase from NEB A type IIS restriction enzyme We used Bsal from NEB Standard cloning materials are needed for upstream and downstream processing of parts We used Q5 HF DNA Polymerase for PCR DH5a E coli for plasmid amplification oligos and constructs from IDT and purification kits from Invitrogen Zymo and Qiagen Cc e r gt e e e e Design considerations A e The sticky ends that differentiate the A B and C pieces must differ by at least two base pairs between the A B and C pieces and must not have GC residues on the ends of the annealing region Avoid more than 2 3 GC pairs in the sticky ends The sequences we used I No are shown in Figure 27 Monomer AB Monomer AB Monomer CA 5 Gain EEEEEE 3 5 QHEEEEEE y mmm 3 5 CGIG aa 3 3 ACAG 5 3 7 EOS ea ERES TCAA 5 Construct AB BC CA 5 ELLE TGTC ERE SEC 2 ci 3 z ACAG J eter CONES 2 TCAA 5 Figure 27 Diagram of individual ICA monomers with corresponding sticky ends The monomers are named after the sticky ends that they possess on the 5 and 3 ends respectively An assembled 3 mer construct is shown but without the initiator terminator or caps gt Fa z D Y Y lt oO cD ar Q e U o A dad ran fad cD Lf Experiences How did you experience working with this cloning method ICA w
24. G Ars C T C Sticky end cutters Enzymes that cut the double stranded target DNA at different Figure Z Recognition sequences of Type II positions creating short overhangs of 1 to restriction enzymes with cut sites in pink T y pe 4 nucleotides so called sticky ends II restriction enzymes either yield A blunt ends or B sticky ends Points of interest Traditional cloning requires a linearized and dephosphorylated vector to prevent self liga tion The vector is linearized by restriction digestion The linear insert needs short complementary overhangs to the vector provided by restric tion digestion Restriction digest with different restriction enzymes leads to directed insertion of the frag ments Over 600 restriction enzymes are commercially available When using a restriction enzyme you should always consider the heat inactivation tem perature as well as the buffer in which the enzyme works Advantages DRACA e Easy to use Need for the respective restriction sites e Easy to troubleshoot Restriction sites cannot occur some e Relatively cheap where else on the vector or gene of interest Quite laborious Cannot change multiple parts in one instance Relatively low efficiency ame e t le Qu c A P E le o0 E ar I Q ar S O imd 10 Frequently Asked Questions e What if my Gene of Interest GOI is not flanked by the desired restriction sites You can always att
25. Introduction Over the past two decades TOPO cloning has become one of the most reliable techniques in the field of cloning The technique s key element is the DNA Topoisomerase I which biologically fulfills the role of cleaving and rejoining the DNA during replication Topoisomerase I from Vaccinia virus cleaves a single strand of dsDNA by specifically reacting with the phosphodiester backbone of a 5 C T CCTT 3 sequence The energy that this reaction releases is then conserved and applied to the formation of a covalent bond between a tyrosine Tyr 274 of the topoisomer ase and the phosphate group attached to the 3 prime end As it cleaves only one DNA strand it enables the unwinding of supercoiled DNA molecules The 5 hydroxyl is able to reverse the reaction that resulted in the initial binding of DNA Topoisomerase I subsequently relegating the single strand Additional to the principle of DNA Topoisomerase I TOPO TA is a one step cloning strategy that is built upon the principle of Taq Polymerase This specific polymerase adds a single aden osine A to the 3 ends of PCR products creating a mononucleotide overhang The TOPO TA kit provides a linearized vector with a mononucleotide thymine T overhang which allows the PCR product to ligate with the vector Figure 39 uononpodu 69 bb ar E Q gt O Points of Interest TA cloning without topoisomerase already had a good reputation as a reliable cloning tec
26. Iterative Capped Assembly Monomers are attached to a growing chain of DNA in an A B C fashion The growing chains are immobilized on streptavidin beads Unre acted chains are capped to prevent them from growing further This image was adapted from Eriggs et al The Steps Iterative capped assembly is similar to Golden Gate assembly which uses unique sticky ends to assemble gene fragments in a specific order Whereas Golden Gate is a one pot reaction with all the pieces ligated simultaneously ICA is a more controlled variation where pieces are assembled one at a time ICA relies on using 3 different versions of the monomer to be assem bled each of which has different sticky ends such that the monomers must be assembled in an A B C fashion This prevents monomers from self ligating Type IIS restriction enzymes which cleave outside of the recognition site are used to generate these In each extension step the next sequential monomer A B or C is added onto the growing chain See Figure 23 Chains that were not extended during the previous extension step are capped using a hair pin oligo that prevents subsequent extension These capped chains re main present in the mixture for the duration of ICA but do not participate in ligation events Each final construct is flanked by a biotinylated initiator oligo which allows immobilization onto streptavidin beads and a terminator oligo These two oligos provide primer annealing sites which ca
27. OT ACC E 3 2 NYU OCU O IN ccs coe sta eocs cs exes cusses 5 3 Table of eo QI I MUERE 7 4 Traditional Orco qo M eR 9 4 1 JManchester OEdZ ue eterne tete ettari serito rir ste Fea reisen ae cines br Ete Eds 12 uS MER vera zia I RRRRRRRNE T5 4 3 Guardia lle n RRRRRURRER 18 5 3A Assembly ene PPP ee 20 SUMUS C te 23 6 Gibson ASSEMDILY cc cccscscesscscsssscscsscsssscsssscscsssscsssscssssssssssscsssscsssscsssscssssssssssscessscsssssssecseseseaceses 26 xo M US cs C60 1 N eerie N nr Te eT Or On 30 57 ari PAS eUT oa EAEE ES EEEE EEE EENES AERIENE REET 33 e e Mm o a 27 E AAA AI N E ee 35 VAM M Fusion Cloning RRRRR 38 sA MEINER 41 8 Iterative Capped Assembly seseeseseseseesesesesesessesesesesssseseseressesesssesersesesessseesesesssesseseseseseesoseseses 44 OL UCLA s 49 9 Golden Gate Assembly seseseeeesesesesssesesesessesesesessssssesesesessesesesesessesesesessosssesesesersesesesessseesesesesess 52 21 NRP UEA 55 2 HE 26 EE A EEEE EEA EEA EEE EEEN EEE EE A EEEE 58 263 VUNE earn EEEE EREE NEEN EAE EAA A ANRE 61 10 Yeast Recombination eeesesesessesesesesessesesesereessseseseseesesesesessesesessseo
28. Points of interests The efficient one pot one step reaction can be carried out efficiently as long as Parts are flanked by a convergent pair of type IIS recognition sequences The acceptor has a divergent pair of recognition sequences for the same enzyme There are no other recognition sites for the enzyme in any plasmid backbones or in any of the parts Overhangs created by the type IIS restriction enzymes are unique 2 Advantages Disadvantages e User defined overhangs yield no scars e There may be one or several internal between the assembly fragments Bsal sites in the gene of interest 1 It is time and cost efficient for large It is less sequence independent than constructs as restriction and ligation are overlap depended methods of assembly performed together 4 5 No PCR or gel purification steps are needed It has no buffer incompatibility as the same enzyme is used 1 Assembly Standards Golden Gate cloning has been cited in the development of several assembly standards and plasmid tool kits These assembly standards prevent the final construct from being scarless but allow for the method to be interchangeable between labs The most widely used assembly standards include uononponu Golden Braid For more information see e Sarrion Perdigones 2011 e Sarrion Perdigones 2013 Modular Cloning MoClo The first step in Golden Gate Cloning is to clone level O modules These are basic parts of genet ic syntax e g promoter
29. The Cloning Guide The first step towards a succesful iGEM Project GOLDEN tte rative ose LTERATIVE cicing CLONING Capped Recambihatiah FUSinhauds 8 A p p F S TON b CAT EXTENSION ITERATIVE shen ay E VORTE E GATE b PCR rim C GOLDEN apped xA S E M B E ONBIWATION 5 E Yos p Nm bl BS ON M Sorten toro FU IN cisson Ww CAPPED Clohihg a GIBSON Es T A i Cu GOL n RECOMBINATION CLONING asimyTraditiaha YE A S T TA 1 AT TERATT VEGON FuSiah E Recambihatiah EXTENSION TRADITIONAL GIBSON Traditighal RECOMBINATION B bSeh VANDERBILTUNIVERSITY NN iGEM Bonn iGEM Manchester Graz iGEM Vanderbilt Carnegie Mellon iGEM UIUC iGEM iGEM Toulouse UCLA iGEM NRP UEA iGEM iGEM Evry iGEM Minnesota iGEM Sydney iGEM York iGEM Pasteur iGEM Stockholm Peace The cloning guide which is lying before you or on your desktop is a document brought to you by iGEM TU Eindhoven in collaboration with numerous iGEM International Genetically Engi neered Machine teams during iGEM 2015 An important part in the iGEM competition is the collabration of your own team with other teams These collaborations are fully in line with the dedications of the iGEM Foundation on education and competition the advancement of syn thetic biology and the development of an open community and collaboration Collaborations between groups
30. The different overhangs must not be complementary The overhangs must not dimerize both homodimerization as well as hetero dimerization They must not be palindromic No more than two of the same bases next to each other They must alwaysend with GC 58 Golden Gate Assembly Genes of interest to insert Primers with Bsal overhangs 5 3 FORWARD 5 CGTGGTCTCNNNNN c g 5 REVERSE 3 GCTGGTCTCXXXXX 5 eu Dsal recognition site eue Future overhang ES Genes of interest to insert 5 CGTGGTCTCN ga XXXXXGAGACCAGC 3 3 GCACCAGAGNNNNN 7 7 XXXXXCTCTGGTCG 5 zm Amplified gene with designed overhangs Figure 32 Overhangs design This example shows Bsal and uses only one gene of interest T he gene of interest can be amplified with primers with Bsal overhangs to enable Golden Gate Cloning Materials During our experiments we used the following Cloning vector with 2 type IIS restriction endonuclease sites flanking a reporter gene Our team used Bsal Inserts with the same designed sites such that the overhangs match those on the cloning vectors Type IIS restriction endonucleases T4 DNA Ligase and T4 DNA Ligase Buffer Geneious software for designing our insert primers and for almost doing anything on virtual DNA Experiences How did you experience working with this cloning method This cloning me
31. This year we aim to develop butyrated glycogen and starch in light of recent research that indicates these forms of starch could prevent colon cancer In the distant future we hope for the possibility of a probiotic to be developed Figure 31 Photo of the NRP UEA iGEM Team From left to Right Richard Bowater Eleftharia Trampari Kieran Rustage Flavia Valeo Leda Coelewij Pilar Moreno Nicola Patron Josh Thody Farhan Mithia Sibyl Batey Mark Banfield Not pictured Mark Riemer Elms Protocol This page contains the protocols used by the NRP UEA team These protocols include e Aone step Golden Gate digestion ligation protocol e Restriction digest protocols IGEM Evry Why Golden Gate The Golden Gate Assembly is a fast and simple method to insert one or several fragments into a vector in a single reaction One of the main advantages is that the overhangs are not de pending on the restriction site such that personalized overhangs can be designed and no scars remain Thus digestion and ligation can be done simultaneously Our team chose to use this cloning method during the iGEM competition because it is com pared to other widespread cloning method cheap fast and convenient single tube reaction Furthermore a lot of people in our lab including our advisors and members of the previous iGEM teams of Evry have experience with this cloning method Design Conciderations A few rules must be followed for the overhangs e
32. ach additional restriction sites with the use of PCR Design complemen tary primers to your 5 ends and add the restriction site of your choice as well as 4 6 random nucleotides to allow the endonuclease to properly bind the DNA How long should I digest my DNA with common restriction enzymes 10 units enzyme are typically enough to digest 1 ug of DNA within an hour at 37 C This time can be reduced or prolonged depending on the respective enzyme used Even though most commonly used restriction enzymes only recognize a specific sequence too long incubation can result in so called star activity meaning that unspecific sequences also get cleaved Do I always need sticky ends You can also do blunt end cloning For example synthesized gBlocks typically come with blunt ends and can be cloned into vectors that were also cleaved with restriction enzymes that produce blunt ends However blunt end cloning typically is less efficient than sticky end cloning and the direction of the gBlock is random Can I use the same sticky ends at both ends of my gene of interest Yes however this way the direction of the inserted DNA fragment is random Two different overhangs allow you to clone your gene of interest in a defined direction Optional PCR Colony PCR Miniprep Sequencing Amplification ABOUT 2 HOURS 1 HOUR results 2 4 HOURS D Ren OVERNIGHT 1 HOUR B Purification Q 30 ys Gel KE Q 2 HOURS Ligation 2 HOURS Transformat
33. agemann Alena Sommer Guido de Boer Mariya Chernyavska Sophia Madler References 1 Thermo Fisher 2015 TOPO Cloning Technolog Brochure 2 Sigma Aldrich 2015 Topoisomerase I from Vaccinia Virus Datasheet 3 Holton T A Graham M W 199 A simple and efficient method for direct cloning of PCR products using ddT tailed vectors Nucleic Acids research Vol 19 No 5 1156 Overlap Extension PCR Introduction Today many different cloning methods are available among several show high efficiency and reproducibility Despite these great attributions most of these cloning methods rely on the usage of at least one restriction enzyme The introduction of restriction enzymes in the parts may cause issues for the overall construct such as restriction scarring Kary Mullis received the Nobel Prize in Chemistry in 1993 for his invention of the Polymer ase Chain Reaction PCR This discovery has accelerated the field of molecular biology im mensely PCR methods have been shown to be eligible for side directed cloning of DNA frag ments into plasmid backbones Several different approaches have been developed in recent years such as TA Cloning Ligation independent cloning and Overlap Extension PCR OE PCR Most of these PCR based cloning methods are very straight forward and easy to apply Their independence of restriction enzymes makes them an interesting alternative to traditional cloning methods OE PCR represents one of t
34. bly PCA kit that can be used for the assembly of multiple overlapping fragments planned to use it as a back up of the NEBuilder Protocols iGEM team York has collected all their protocols on the following page These protocols were used to perform their own Gibson Assembly and it shows their specific details on lab work H10 WID SC tm EC z Q UD lt ar o n QO 36 Experiences How did you experience working with this cloning method The Gibson Assembly worked reliably when the DNA fragments and vector were highly purified and in the proper molar ratios In our experience small levels of contamination eg Non specific amplification from genomic DNA or colony PCR that escape PCR purification can cause downstream issues in the cloning of large protein coding genes and growth im peding proteins What was the most difficult task We designed a synthetic operon that contains 4 protein coding cistrons We wanted to be able to assemble several interchangeable variations of the operon with some deleted protein domains and a variant without one of the proteins and to be able to do this with the least amount of gBlocks We had to design short adapters to link each gene fragment flanking the domain deletions and incorporate restriction enzyme sites surrounding the above mentioned protein Did the cloning method work as expected The cloning of fragments up to 2 1 kb worked as expected from first a
35. buffer to use restriction or ligase buffer as the protocol will differ depend on the buffer Bpil is required to make level O constructs 3 To make level O parts using the ligase buffer the following was added a 100 200ng of acceptor plasmid plasmids containing each module to be inserted in a 2 1 ratio of insert and acceptor b 1 5uL of T4 ligase buffer with 200 units of the T4 DNA ligase c 1 5 ul of Brovine Serum Albumin 10x and 5 units of Bpil They are then put in the following conditions 20 seconds 37 C 3 minutes 37 C 4 minutes 16 C X26 5 minutes 50 C 5 minutes 80 C 5 minutes 16 C To make level 1s the type of buffer must again be decided Using the ligase buffer will fol low the same conditions as the level O but Bsal will be used To make level 2s repeat step 3 with the products of step 4 optional Use 5 ul of the one pot dig lig reaction to transform electrocompetent E coli cells Select positive clones using LB agar with appropriate antibiotics Decideon the primers used for amplification and or sequencing 7 Wad VAN ddN GS Experiences e How did you experience working with this cloning method The use of this cloning method in comparison to different methods used by the other members of the team showed an impressive efficiency with hardly any problems What was the most difficult task Designing the level O constructs is the most difficult task After the level Os are made and wor
36. cted Target vector for integration PCR purification Kit from Qiagen PCR machine with high performance e g Eppendorf Mastercycler nexus Sequencing Service such as Sanger Sequencing e g GATC Biotech Optional DpnI Restriction Enzyme to lower colony background e g NEBG OMNAMAWNE e om OmpR Protein biomarker Mici 3 ib D wa LLL MAII e 07 WIN S A TTO P0 CHR aaa uSESMuEuaEEMEuuuEHuuuuuuuuuuu Figure 44 One of the example signaling pathways used by iGEM team Stockholm The Overlap Exten sion PCR has to be performed to do further reasearch on examples as pathways Protocols The following QR code will direct you to the protocols used by our team These protocols have been based on other protocols already existing for OE PCR iGEM team Stockholm Our team consists of eighteen undergraduate and overgraduate students from Karolinska In stitutet and KTH Royal Institute of Technology who are coming from five different educational backgrounds and are united in the vision to find new ways for early disease detection Hence in 2015 we were exploring a novel way to detect selectively and sensitively biomarkers in dif ferent body fluid samples A new class of chimeric receptors derived from the strong peptide binder the Affibody molecule and the bacterial membrane receptor EnvZ should sensitize bacteria and trigger a signal amplification cascade
37. ction enzymes you have to make sure to use an optimal buffer for both enzymes The Double Digest Calculator allows you to quickly find the right buffer Enzyme Finder by New England Biolabs This tool gives you a nice overview of all commercially available restriction enzymes as well as their recognition sequences and other properties of interest ApE ApE is a nice freeware plasmid editor that allows you to display your plasmid with its restriction sites do in silico cloning or simulate aga rose gels after certain restriction digests IGEM Manchester Graz Why traditional cloning Standard molecular cloning is the main technique often used by iGEM teams It has the ability to transfer genes from almost any organism into a host such as E coli Manchester chose this tried and tested method as our project is relatively simple in its requirements for recombinant gene expression Furthermore here at the Manchester Institute of Biotechnology we already have much of the equipment required for traditional cloning enabling us to insert plasmids and grow up E coli to a large volume both cheaply and efficiently Materials Vector NTI by Life Technologies to design our constructs SnapGeneQ to design subcloning PCR primers gBlocks and custom oligos by Integrated DNA Technologies B pIrcHis2 by Invitrogen S pCDF 1b by Novagen O Ncol AvrII and HindIII restriction enzymes by NEB T4 ligase by NEB c aa z E Design concid
38. ecessary to have three antibiotic markers Can t I just use two instead each insert has antibiotic A while the backbone has antibiotic B We think this would work perfectly fine The only disadvantage would be that it s more difficult to tell your inserts apart if you are troubleshooting later on I m assembling two Biobricks together Should I use 3A Assembly or traditional cloning This really depends on what parts you re using Many iGEM parts are in backbones with chloramphenicol resistance such as PSB1C3 If you want to put together two parts from PS B1C3 and have the assembled product still have a PSB1C3 backbone then you ll probably want to use traditional assembly However if you have different backbones for your com ponent parts and your desired construct then you may find that 3A Assembly is quicker and easier than the alternatives Is it necessary to test the assembled construct for accuracy In theory only the correct constructs should remain once you grow your constructs on the appropriate antibiotic However a quick double digest and diagnostic gel is always a good step to take to confirm that your construct is the correct one gt d a lt lt S 22 Useful additional information amp resources iGEM Academy has published a video which walks you through the pro cess of performing a BioBrick 3A Assembly The video includes the proto cols for digestion amp ligation If you are interested in a recent pa
39. ents on hairpins cross di mers and G quadruplexes You don t want secondary structures in your overlaps If neces sary alter the dsDNA fragments using codon optimization Check all dsDNA fragments on prohibited restriction sites PstI EcoRI Xbal and Spel These are required for BioBricking and thus forbidden to use within the fragments Check overhangs and the T with the SnapGene T calculator Order as gBlocks or PCR your dsDNA fragments Experiences How did you experience working with this cloning method We had a few problems starting with Gibson Assembly our foolishness led to us using the wrong primers for instance In the end we became very familiar with the method The assembly itself is fairly easy and very intuitive What was the most dif ficult task Designing of the gBlocks was the hardest part for our project Especially the overlapping se quences required a lot of checking and fine tuning Moreover we had some setbacks using Gibson Assembly and troubleshooting is also fairly difficult since many steps are integrat ed into a single tube reaction Did the cloning method work as expected Yes the cloning method worked as expected in virtually all assemblies As mentioned we had a little trouble starting with Gibson Assembly when we used the wrong primers to linearize the vector Strangely so the vector had closed on itself such that we could find colonies on our agar plates It was not until s
40. equencing when we discovered our mistake What was the biggest achievement using this cloning method The biggest achievement was to design gBlocks for the Gibson Assembly which would work as desired combined with the fact that there are some requirements for the gBlocks secondary structure GC content and T_ are some issues to take into account and when this needs to be done for multiple gBlocks this may be challenging What would be your tips and tricks if other teams are going to use this method First of all it is important to get some background on the several cloning methods available nowadays A guide like this can help since we know that all the literature available may be overwhelming in the beginning Second list all the requirements for your optimal cloning method and compare them with the cloning methods available All methods do have their advantages and disadvantages so choose one which suits the needs for your project per fectly Once you have settled for a cloning method make sure to read out available litera ture and especially material from cloning kits supplier Then entering the lab will become child s play C Ti Ke C Er Ou ay le lt D OO ice iGEM TU Eindhoven Our team consists of eleven undergraduate students from Eindhoven University of Technol ogy at the faculty of Biomedical Engineering This year we are developing a modular mem brane sensor based on click chemistry and aptamers
41. er primers for your OE PCR We recommend you to take one or two days to sit and try different variants of the primers and to play the scenarios through in silico using Snapgene 76 Overlap Extension PCR e Did the cloning method work as expected It worked as mentioned very well for us and as expected with the one construct which contained repeating sequences we had really hard times synthesizing it However it is doable e What was the biggest achievement using this cloning method We cloned within a week all three genetic chimeric constructs into a functional plasmid with which we could start precise characterization right away We could catch up on time that we lost in prior parts of the project e What would be your tips and tricks if other teams are going to use this method o Take you time and prepare your OE PCR carefully Think about how many mismatches you can accept in the 3 area of your primer and whether the 5 elongation part could par tially hybridize in other parts of the plasmid o Use DpnlI and the PCR purification kit to increase your yields especially when having several fragments that you want to clone together Materials The Q5 High Fidelity DNA Polymerase Master Mix offered by New England Biolabs Snapgene especially its Overlap Extension PCR action for in silico simulation Appropriate primers to extend insert with flanking regions Insert sequence vector from which insert sequence is extra
42. erations 1 You need to know the restriction enzyme sites RE available for sub cloning in the parent A vector multiple cloning site MCS and the destination vector MCS RE sites in the parent MCS m should either be common with or compatible with destination MCSs The RE site should also not be within the target gene Double digests are performed on parent and destination vectors If a common buffer is used make sure to use a buffer where enzyme activity is at least 7596 If the two enzymes are not compatible either sequential digests longer incubation in buffer or addition of more en zyme can be carried out Destination vector is dephosphorylated to prevent self ligation by removal of 5 phosphates of linearized vector Calf intestinal alkaline phosphatase CIAP is most commonly used for dephosphorylation and can be used for REs that produce 3 overhangs 5 overhangs and blunt ends CIAP is removed via gel electrophoresis direct purification or gel isolation using DNA purification systems Shrimp alkaline phosphatase is an alternative and can be heat denatured removing the need for purification Gel purification can be performed to remove uncut or partially cut destination vectors Negative controls with self ligated vectors can indicate the proportion of uncut or self ligat ing vectors in the final sample Control for ligation reaction can be set up It would contain all components of ligation mix except the gene Both the control
43. eted for the 5 overhangs This problem was eventually overcome by Gibson by swapping the T4 Polymerase for a T5 Exonuclease which digests DNA from the 5 to the 3 end 2 This change enabled Gibson Assembly to become a very efficient method nowadays up to six dsDNA fragments can be assembled within a single Gibson Assembly reaction Points of interests Gibson Assembly requires linearized vectors and dsDNA fragments The vector can either be linearized by restriction digestion or PCR Gibson himself used PCR in his revolutionary paper which was published in Science 1 The dsDNA fragments need to have complementary overhangs with adjacent fragments This is needed to anneal the fragments in the right order during the Gibson Assembly The dsDNA fragments can be directly ordered at a supplier or generated by PCR The overlaps should be clear of secondary structures and range from 15 80 basepairs The overlaps should be unique This makes Gibson Assembly difficult albeit not impossible for repetitive sequences Gibson Assembly 5 y Exonuclease nibbles back the 5 ends y I ay DNA Polymerase extends the 3 ends DNA Ligase seals the nick Figure 12 Overview of the one tube assembly reaction First the exonuclease activity nibbles back the 5 ends of the dsDNA fragments Second the DNA fragments anneal due to the newly exposed com plementary ends Third the DNA Polymerase extends the chewed back 3 ends Finally the DNA Li
44. g ICA is shortened UCLA iGEM UCLA iGEM is working on creating and expressing silk from customizable spider silk genes which contain many repetitive sequences assembled sequentially We thought that ICA would be perfect for our project because it allows us a high level of control over the construc tion of our silk genes THEE LA Figure 26 UCLA IGEM Team in front of Boyer Hall which houses the Molecular Biology Institute and the UCLA Department of Energy DOE Institute Back Row Left to Right Carter Allen Tyler Lee Tristan Joseph Vinson Lam Middle Row Left to Right Michael Cheng Phillip Nguyen Nithin Dharmaraj Fasih Ahsan Front Row Left to Right Megan Satyadi Jessica Huang Olivia Cheng Protocols e Making MaSp ICA Monomers e CA Oligo Sequences e ICA Preparation Golden Gate Assembly Introduction Golden Gate Cloning was first developed in 2008 It was devised in order to make a technol ogy of cloning that was fast efficient and did not leave cloning scars 1 This cloning tech nique uses type IIS restriction enzymes and T4 DNA ligase This enables assembly of multiple DNA parts in what is often referred to as a one pot one step reaction see Figure 29 This is achieved because type IIS restriction enzymes cut outside of the recognition sequence so the overhangs produced by the enzyme can be user defined This characteristic allows multiple parts to be assembled in pre determined order and orienta
45. gase seals the nick yielding a continuous DNA plasmid This image was adapted from NEB Advantages Disadvantages e Gibson Assembly is a method which ena e Gibson Assembly requires meticulous bles directional cloning Gibson Assembly is very rapid and ef ficient multiple fragments can be com bined in a single tube reaction Possiblity to assemble large gene frag ments and generation of gene libraries Gibson Assembly is a seamless metho It does not yield any scars which cloning methods that rely on restriction en zymes do have design of your experiments Gibson Assembly is very difficult to troubleshoot If your assembly does not work youd better have a back up plan at hand Gibson Assembly fragments are virtu ally non interchangeable the fragment overlaps are very specific causing trou ble when parts need to be switched out ne FE m Q Qu c A rE j e le tm EC z Q UD lt ar o n QO 28 Further applications of Gibson Assembly e Site Directed Mutagenesis see Figure 13 Gibson Assembly can be used to make rapid changes to DNA fragments including substitu tions deletions and insertions see Figure 13 To use Gibson Assembly for mutagenesis the desired changes are introduced into primers such that fragments are generated carrying the mutations in their overlapping sequences To modify a DNA sequence in this way two primers are required per mutation both car
46. gland Biolabs e SnapGene s and especially its in silico cloning tools see Figure 14 Q e The Q5 High Fidelity Polymerase Master Mix offered by New England Biolabs e GeneRunner to check the overlaps for hairpins and dimers e NUPACK to verify cross dimers between the different overlaps ES e QGRS Mapper to generate information on possible G quadruplexes 45 C 61 C 62 C 60 C hon nn Figure 14 Melting temperatures of overlapping sequences play an essential role during Gibson As sembly ensuring specificity and increasing efficiency Various in silico tools exist to simulate the Gibson Assembly and corresponding melting tempteratures This image was adapted from SnapGene Design Conciderations Choose a vector appropriate for the project you are working on Make a decision on how to linearize the vector prior to the Gibson Assembly reaction This can be done using restriction enzymes or PCR with appropriate primers In the latter case digestion of the PCR template by DpnI can be done to minimize background colonies Implement all coding sequences that you will use as dsDNA fragments in seperate Snap Gene files These fragments can have a maximum size of 2000 bp Create a 15 80 bp overhang to all adjacent dsDNA fragments This differs per protocol so it is wise to read the manual provided with your kit Check about 100 bases on each 3 to 5 end of the dsDNA fragm
47. hat temperature and for how long it has to be inactivated In addition to the digestion the ligation can vary in times and temperatures depending on the size of the fragment and the type of overhang Traditional cloning works well when each parameter is optimized for the enzymes and sequences being used What was the most difficult task The most difficult task is determining the proper enzymes to use Their restriction sites need to be at the right position there needs to be enough space adjacent to them for the enzyme to cut and many other factors described above need to be taken into consideration Did the cloning method work as expected We have had great success using this cloning method However even experienced labs will occasionally have difficulties using this technique Despite this when optimized this meth od of cloning should have a success rate of well above 9096 What was the biggest achievement using this cloning method We were able to take pUC19 a common plasmid and turn it into a yeast genomic integration vector that also has bacterial expression This was done by integration of two gBlocks using BamHI Clal and KpnI sites Then the plasmid was made biobrick compatible by the excision of illegal sites and replacement with short dsDNA formed by annealing oligos which is fast er and cheaper than using site directed mutagenesis What would be your tips and tricks if other teams are going to use this method Spend time
48. he most interesting and most straight forward PCR techniques when it comes to cloning methods The basic principle can be described as a two step meth od 1 Synthesis of your insert fragment with corresponding flanking regions to the insertion location on the vector on both sides the 5 and 3 end respectively of the insert and 2 side di zi rected insertion of the flanked insert into the target plasmid This approach is also described as z circular polymerase extension cloning In this guide we want to introduce the general princi c ple of OE PCR their advantages their limitations and tips about how to improve your OE PCR c cloning method S Fragment elongation for each part you wish to combine N R Direct integration of elongated fragment into target vector ZZ x lt _ lt Figure 42 Traditional OE PCR for synthesis of recombinant fragments In this traditional usage of OE PCR you would need to use PCR amplification to isolate the 5 and 3 fragment as well as the middle piece connecting the two fragments By extending each of these fragments with 25 27bp of the corre sponding neighboring sequence you can easily assemble different parts together This approach repre sents a very powerful tool to create new chimeric biobricks Points of interest Flanking regions should be 25 27 nucleotides long at each end Prior linearization of the plasmid can help to promote better assembly of insert and vector The in
49. his can easily be done by using gBlocks and yeast recombination for only colonies that have the selection marker should survive For more information on yeast plasmids and selection markers check out the addgene piece on yeast vectors iGEM Minnesota 2015 We are a team of five undergraduate students from the University of Minnesota Twin Cities This year we are studying the use of viral 2A sequences in multi enzyme biosynthesis We have developed a mathematical model for optimal gene order 68 Yeast Recombination Materials Growing the yeast e Auxotrophic yeast strain ex CEN PK2 e Yeast extract peptone dextrose YPD plates Yeast Recombination e Plates with necessary compounds yeast otherwise cannot make e 1000 2500 ng linearized vector that provides the gene required for the yeast strain ex pESC URA gBlocks offered by IDT 50 PEG 3350 1M Lithium acetate LiOAc 10 mg mL carrier DNA Lyse Yeast for Colony PCR Screen e 2M Sorbitol e 1M K3PO4 e 20mg ml 20T zymolyase Figure 37 Team minnesota from left to right Nicholas Nesbitt Andrea Willgohs Tanner Cook Sarah Lu cas and Patrick Holec References 1 A Hinnen J B Hicks G R Fink Transformation of yeast Proc Natl Acad Sci U S A 75 1929 33 1978 2 T L Orr Weaver J W Szostak R J Rothstein Yeast transformation a model system for the study of recombination Proc Natl Acad Sci U S A 78 6354 8 1981 3 H Ma S Kunes P
50. hnique as it was first used in 1990 by T A Holton 3 TOPO TA technology is trademarked by Thermo Fisher making them the only supplier Because of this the available vector types is decreased to 2 PCR products from primers with 5 phosphates DO NOT ligate in the vectors Post amplification with Taq Polymerase can create the necessary A overhangs if your PCR product contains blunt ends gt 3 X 3 S Figure 39 pCR2 1 TOPO pCR2 1 comes with a selective LacZ marker that re mains functional if the plasmid relegates with itself Successful ligation of PCR product in the pCR2 1 vector disrupts the lacZ gene which results in white colonies Advantages Specific binding of PCR product to vector due to overhangs As if 5 minutes wasn t already fast TOPO TA can produce successful ligations in 30 seconds o EcoRI sites flank the PCR product insertion site in both vectors for easy excision of inserts s Selection of cells is based on antibi otic resistances Amp and Kan and LacZ ccaB screening Taq amplified PCR Product Figure 40 pCRA TOPO pCR4 comes with a LacZ ccdB marker of which the lethal E coli gene ccdB gene is fused to the C terminus of the LacZ gene Ligation of PCR products disrupts the fused genes expression allowing growth of the transformants Disadvantages TOPO TA is expensive starting at 209 for 10 reactions o TOPO TA is not directional al though TOPO directional cloning k
51. hough there are new technologies on the rise with independent scientists or even companies on the inter net currently the most reliable option is Invitrogen s Custom TOPO Adaptation Service which allows you to send in a glycerol stock of E coli with your plasmid of which a TOPO vector will be made uononponug Usefull aditional information amp resources e General overview of TOPO cloning Invitrogen life technologies has compiled the advantages of TOPO TA Cloning over traditional cloning Of eit i m within this folder Moreover it describes extensively how TOPO fre US TA Cloning works CT ATE kh Custom TOPO Adaptation This powerpoint presentation by ThermoFisher gives a detailed description of TOPO TA Cloning further applications of TOPO TA Cloning Topo TA Cloning IGEM Bonn Team iGEM Bonn Our international team of students comes from a variety of biological studies at the University of Bonn and the Hochschule Bonn Rhein Sieg We research the recycling process of the most common natural resource paper As synthetic biology offers a novel clean and even more efficient approach compared to common chemical procedures we aim to facilitate and contribute to innovation Figure 41 Team photo of iGEM Bonn behind the LIMES Institute Top row from left to right Benedikt Holbling Balthasar Schlotmann Ashwin Shah Pavel Ryzhov Jan Hansen Niklas Schmacke Bottom row from left to right Katrin Ciupka Max Schelski Cathleen H
52. i Transformation Q 2 HOURS Skip day 3 and wait for your results Plate culture OVERNIGHT Note this alternative is quicker if no further amplification of the new construct is needed library Figure 30 General timeline of Golden Gate Cloning Optional or alternative steps are highlighted It takes about 3 days to obtain transformed colonies when using E coli This image was made by iGEM Evry NRP UEA IGEM Why Golden Gate In 2014 the NRP UEA iGEM team did the majority of the cloning using Golden Gate Assembly They also submitted RFC106 that defines standard overhangs for plant parts We hope to repli cate their successes this year An important aspect in our project is to test multiple parts in plants Not only does Golden Gate allow us to rapidly and efficiently test several of these parts together at once it also gives us more ease when inserting our plasmids into plants With experience in this form of cloning from previous years as well as professionals who regularly use Golden Gate Assembly to guide us we feel confident in using it again this year Materials Restriction Enzymes Bsal NEB and Bpil Thermo Fisher and buffer T4 DNA Ligase NEB and buffer NanoDrop Electo competent Cells ElectroPorator Method We have done our cloning according to the TSL protocol which is as follows 1 Add ATP to the buffer that will be used as it is required for the T4 ligase to work 2 Decide which
53. ignificantly lengthen the process a Asked Questions How long does ICA take Depending on the length of the final construct and ligation times ICA can take 3 5 hours From our experience ICA for a 12 mer construct takes 3 hours from placing the first mono mer onto the beads to eluting the final construct e Arethere other Type IIs restriction enzymes that can be used instead of Bsal Other Type IIs restriction enzymes exist such as FokI These can certainly be used and the resulting constructs should be made to accommodate the new recognition site Most Type IIs restriction enzymes cleave DNA so that there is a 4 base pair sticky end If another enzyme is chosen that leaves a different length sticky end this difference should be taken into consideration particularly if the final DNA assembly is a coding sequence e Arethere any special considerations when designing the A B C sticky ends Yes the A B C sticky ends should be unique and should not be able to bind to each other When working with four base pair sticky ends there should be at least a two nucleotide difference between the A B and C sticky ends Finally there should be no more than 2 3 GC pairs in each sticky end and no GC residues on the terminals of the sticky ends p Fa z D Y Y lt O i ar Q e UO e A dad ran fond cD le 0 JT e Howmany pieces can be assembled using ICA Our team has as of August 2015 assembled up to 17 DNA pieces
54. in vitro which we now know as traditional cloning or DNA Recombinant Technology 1 This funda mental discovery by Cohen and Boyer paved the way for biotechnology and synthetic biology as we know it today But cloning has not stood still over the last forty some years Many efforts have been undertaken to improve upon the traditional cloning methods as described by Cohen and Boyer These efforts have resulted in numerous different well known cloning techniques including standardizations as 3A Assembly amp MoClo Research to diversify upon the available cloning methods has moved on apace over the last ten years These research efforts have resulted in cloning methods vastly different from traditional cloning Currently cloning methods have been described which are independent of ligation in dependent of restriction enzymes and even cloning methods independent of the use of a chassis 2 These newly developed methods have also found their way into iGEM providing viable al ternatives to the seemingly ancient method of 3A Assembly Previous Cambridge teams have for example pioneered Gibson Assembly Freiburg has introduced iGEM to the Golden Gate Stand ard and Lethbridge familiarized iGEM with ligation independent cloning Recently many companies active in biotechnology have begun to offer iGEM teams with prod ucts ideally used in combination with these novel cloning methods Integrated DNA Technol ogies gBlocks are ideal for Gibson Assemb
55. ion 5 2 HOURS Figure 3 Schedule of traditional cloning Traditional cloning starts with PCR amplification of the insert if there is too little available Next these parts are digested with the appropriate digestion en zymes Based on the fragments which are removed from the vector or inserts the parts can be purified through either gel extraction or PCR purification The vector and insert can then be ligated into the new vector The vector can then be transformed for plasmid amplification Further applications e Constriction of PCR Product Library When creating a library of PCR products other methods of assembly would be far too com plicated and take too long Blunt ligation solves that problem through the use of the pJET plasmid The product generated by Pfu DNA polymerases generates a blunt end which can then be ligated with the pJET vector which is also cut with a blunt end Alternatively when using a Taq DNA polymerase there is an adenine base that is added to the 3 end of both strands creating a short overhang The TOPO backbone is then used for ligation which has a complementary thymine base at the 5 end of both strands The only issue with these meth ods is that directionality cannot be specified so insertion is random and can only be deter mined by sequencing e Short dsDNA Insertion by Annealing Oligos Single stranded oligonucleotides can be ordered and then annealed very easily using a sim ple thermocycler program in
56. its are available Limited choice of vectors Possible applications of TOPO TA cloning e Subcloning As confirming successful terminal restrictions are hard to confirm on PCR fragments pCR2 1 TOPO offers 15 convenient and validated restriction sites flanking the region of insert Gel electrophoresis would subsequently confirm a successful digestion Sequencing pCR4 TOPO contains 4 priming sites T3 and T7 M13 Reverse and M13 Forward 20 and as the cloning kit comes with the associated primers it enables the user to verify that the genes are ligated in the correct orientation Frequently Asked Questions What if our PCR products only have blunt ends Blunt ended fragments can still be post amplified by incubating them with Taq Polymerase to acquire the necessary A overhangs It is best to do this post amplification right before you intend to TOPO TA cloning e Canlusea DNA Polymerase mixture containing both Taq Polymerase and a proofreading polymerase for TOPO TA cloning It is possible to use a pre mixed polymerase mixture as long as the ratio between Taq Poly merase and proofreading polymerase is 10 1 Is it possible to clone our gene directionally Aside from TOPO TA Thermo Fisher offers multiple other TOPO cloning kits for longer PCR fragments 3 10 kb blunt ends and directional cloning Are there alternative TOPO vectors than those of Thermo Fisher Unfortunately Thermo Fisher is the only supplier of TOPO vectors Alt
57. k it s a simple one pot one step reaction Did the cloning method work as expected The sequence analysis suggests that the cloning of our constructs worked as expected The fluorescent imaging of our first set of constructs reaffirmed this What was the biggest achievement using this cloning method Obtaining our first successful results using Golden Gate Cloning The confocal microscopy imaging was the final indication our cloning had worked What would be your tips and tricks if other team are going to use this method First it is important to know that Golden Gate is the most appropriate form of cloning for your project After that an important piece of advice would be to follow protocols closely Majority of the time mistakes happen when shortcuts are made ES QD z OD i Y lt i T a Q ci i i le O NO Lf Transfection of plants Golden Gate Modular Cloning MoClo toolboxes have been developed for the transfection of plants These make efficient transfection of plants possible iGEM NRP UEA is the only team featured within the guide focusing on transfection of plants and has used MoClo to make them generate resistant starch It is thought that high dietary intake of resistant starch imay reduce colon cancer and inflammatory bowel disease The NRP UEA Team Our team is made up of 6 undergraduate students from University of East Anglia along with 3 PhD students and 3 supervisors from Norwich Research Park
58. lls and analyzed using colony PCR to select the correctly assem bled vectors Introduction Back in 2004 Daniel Gibson and his team undertook the effort to assemble an entire bacteria genome from scratch the Mycoplasma genitalium genome This posed a monumental chal lenge since the genome was far too complex to be assembled by sequential restriction enzyme mediated cloning known as traditional cloning 1 To assemble the genome more efficiently he started with a two stage approach involving three enzymes a 3 5 exonuclease a Tag DNA Polymerase and Taq Ligase The first step involved a 3 5 exonuclease which initially nibbled back the ends of the complementary ends of the DNA fragments which needed to be inserted in the vector After a short incubation the reaction was heat inactivated and then cooled to anneal the newly exposed complementary ends The second step features the polymerase and ligase which respectively fill the gaps between the newly annealed complementary ends and seal the nicks producing a single continuous DNA strand The key to success of the Gibson Assembly method appeared to be ligation which enabled Gib son and his team to build larger DNA fragments and even an entire genome Although they reached their primary goal some tweaking on the Gibson Assembly method was needed The biggest problem during the development of his new technique was the chew back step since the 3 5 exonuclease and the DNA Polymerase comp
59. ly and New England Biolabs provides iGEM teams with a choice in cloning kits including kits for BioBricking Golden Gate Assembly and Gibson Assembly Novel cloning methods have thus started to play a major role within the iGEM competition and we think that they will become even more important in the future However as a new iGEM team still unfamiliar with DNA Recombinant technology let alone those newer cloning meth ods we couldnt see the forest for the trees In the end we settled for a combination of Gibson Assembly amp BioBricking which will probably do just fine But to enable future iGEM teams to make a more informed choice on assembly methods we thought of compiling a cloning guide We cannot hope that the cloning guide can walk iGEM teams through all the ins and outs of mo lecular cloning Sambrook and Russel do this in Molecular Cloning a laboratory manual widely regarded as the bible of biology but also three volumes thick However we do hope the cloning guide can serve as a stepping stone for future iGEM teams in finding their cloning method of choice To be able to compile a cloning guide which can serve as a stepping stone for future iGEM teams we rely heavily on collaborations with other teams Therefore we have reached out to many other iGEM teamson this guide who have hands on experience with many different clon ing methods and have tinkered with their protocols Kind regards TU Eindhoven aloe COMENTS Te
60. mbly strategy featured a combination of Gibson Assembly amp Traditional Cloning cL Protocols A direct link to the protocols of the Manchester Graz team The protocols include digestion ligation and amplification steps which the team has performed with this specific cloning method iGEM Manchester Graz Our team consists of six students from the University of Manchester and six students from the Graz University of Technology Being an inter European team has given us the chance to develop a project with two interlinking parts We are developing a novel drug delivery system for L DOPA and dopamine alongside a multi dimensional regulation system for protein expres sion with the future potential for implementation as a self regulated one course treatment for Parkinsons disease d A 2 E i oc See met E i i ME PAG ry LA h Ji I Figure 5 Team photo of the inter European GEM Manchester Graz Rachel Stirrup Magdalena Kurteu Maria Imran Priyanshu Sinha Aaron Gretton Iaroslav Kosov Christoph Schilling Markus Hobisch Mar tin Senekowitsch Peter Kusstatscher Kerstin Stadler Maria Hulla Melanie Ballach IGEM Vanderbilt Why traditional cloning This cloning method allows a lot of flexibility there are no illegal restriction sites that have to be avoided since any restriction enzyme that has a recognition site in the sequence can be used Fu
61. mes 3 https www neb com tools and resources usage guidelines cloning guide uOT PW edeures 6l 3A Assemb Introduction The story of 3A assembly is tied up with the story of BioBricks themselves All BioBricks con tain a prefix and a suffix standardized sequences of DNA about 20 base pairs long that can be cut by specific restriction enzymes 1 This feature is what allows BioBricks to be easily com bined with one another While the aptly named traditional assembly remains the most fundamental method of cloning BioBricks 3A assembly offers a useful alternative 3A stands for 3 antibiotic which refers to the different antibiotic present in the backbones of each of the parts to be combined Although 3A assembly cannot be used in as many cases as standard assembly for example it will not work if you wish for your final construct to have the same antibiotic resistance as its component parts 3A assembly offers some distinct advantages over standard assembly For example it has a higher success rate and its products do not need to be gel purified 2 In contrast to standard assembly in which one part is cut from its backbone and ligated into the backbone of another part 3A assembly involves cutting both parts from their vectors and ligating them with a linearized backbone However as figure 8 shows 3A assembly follows the same principle of cutting the BioBrick prefix and suffix with restriction enzymes then ligating them
62. most of the problems we had at first What would be your tips and tricks if other teams are going to use this method We would tell you to read all you can find about the Gibson Assembly and to speak with researchers who use it on a regular basis if you have the opportunity to do so Materials For the design of our overlapping sequences we used the software Geneious which of fered free licenses to all of our team members We had to order some of our fragments mostly the intersequences We used the company MWG EurofinsQ for the synthesis of our oligos and primers We used the NEBuilder cloning kit provided by NEB our sponsor Gibson assembly Master Mix TaKaRa Ex Tag polymerase for PCR amplification u aoypu NL WID Designing our Operon We are assembling an operon of thirteen genes measuring from 460 to 3140 basepairs In or der to find the best possible combination of those genes we designed our operon in a way that we could use Cre Lox recombination to test the different combinations Therefore we had to insert intersequences between each gene Those intersequences contain a Lox sequence and are built as such However there is still a high number of possibilities about six billions so we decided to divide the genes into four clusters The clusters have intersequences containing the Lox sequence for one Cre and the intersequences between the clusters contain the specific sequence for anoth er Cre This
63. n of modular repetitive sequences It is Golden Gate Assembly or Gibson Assem thus very good with these sequences bly when constructing non repetitive Assembly of repetitive gene sequences is sequences difficult with more conventional tech Difficulty of using ICA as a routine niques involving PCR assembly method arises from needing The sticky parts used in ICA are very to introduce the required sticky ends interchangeable and it is possible to through end extension PCR assemble anything as long as the proper sticky ends are in place ICA can be designed to be a scarless as sembly but it does not necessarily need to be ICA vs Golden Gate e Both ICA and Golden Gate Assembly rely on the use of type IIS restriction enzymes to cre ate compatible sticky ends that can no longer be cleaved after being joined ICA and Golden Gate have the ability to assemble multiple pieces by designing unique sticky ends However Golden Gate Assembly is limited in the number of parts that can be assembled within a single reaction due to the combinatorial limitation of sticky ends ICA is like a controlled Golden Gate Assembly where one piece is assembled at a time rath er than all simultaneously This removes the need for many unique sticky ends and allows for the reuse of sticky end sequences as well as increasing the possible length of the final construct However the tradeoff is that each piece must be assembled individually which can s
64. n be used to amplify the sequence using conventional PCR see Figure 24 The capped chains are not amplified in this PCR as they lack the terminator oligo EN CES cE5 NEN Assembly into a vector Figure 24 After the polymer has been constructed on the beads they are eluted from the beads The eluate is used as a template for PCR to amplify the construct Only complete constructs that contain initiator and terminator are amplified Capped oligos are not amplified as they lack the primer binding site n le Qu c A F E le u pe Fa Qo Y Y lt oO i ar Q e Q QD A dad ran hel cD NO JT Points of interests e The ICA monomers are generated using type IIS restriction enzymes e g Bsal which cleave asymmetrically outside of the recognition site It is of the utmost importance that your DNA does not contain these sites elsewhere particularly within your monomer ICA is the only method within this cloning guide that assembles DNA constructs on a solid substrate The initiator terminator and capping oligos are prepared ahead of time by mixing the rele vant oligos and ramping down from 95 C to form the working oligos Bsal Q 4 Bsal o amp Z9 lt GGTCTC CCAGAG CTCTGG Bsal digestion EH a m M Figure 25 Parts are generated by digestion of the monomers by the Bsal restriction enzyme Even though the core monomers remain the same darker blue the
65. nd side of the light blue overhang 5 Design primers for PCR linearization of the vector such that they are amplified from a few base pairs upstream of the desired cut sites of the other vector 6 Double check that all overhangs overlap and are in the correct overhang type of 3 or 5 In other words reconstruct the final recombinant vector after drafting your designed inserts and vector 7 In the thermocycler the 37 C cycle is for Bsal digestion and the 16 C cycle for DNA ligase to join the overlapping overhangs together AsupAs JA3O T9 E QD z o WY Y lt i T a Q c i i le O N NO Results amp Tips We discovered that Golden Gate gives mixed results 0 For one of our constructs it successfully joined three 1 1 5 kbp inserted into digested pSB1C3 using the above method o In the other construct while the transformation gave positive results it was discovered that instead of ligating two 2 kbp inserts into pSB1C3 it ligated a 400 bp insert in it which is the subject of much mystery and speculation o Design inserts such that they contain the full ORF not divided between two inserts as this can make the design of overlapping regions complicated o Note that the Bsal cut sites are removed after Bsal digestion hence the ligated inserts will not be digested again and the Bsal will go on to digest inserts The method works well provided that the following are implemented Concentrated T4 DNA ligase
66. ng the lox sequences into one cluster We looked at all the possible assemblies and finally settled on Gibson Assem bly because it was the fastest method of assembly Experiences How did you experience working with this cloning method We had some troubles because of the design of our inter sequences The lox sequences were actually too close to the overlapping sequence This caused them to be exposed by the exonuclease and to hybridize together This caused hindering the polymerization on our Gibson What was the most difficult task The most difficult and challenging task for us was to design our operons and the way we would assemble all of our fragments We also decided to do a linear Gibson Assembly so we didn t assemble our fragments in a plasmid in order to do a bacterial amplification We decided to use PCR amplification on our assembled clusters Using this method required us to do more research and solve more problems Did the cloning method work as expected We had a few troubles when we started assembling our clusters but we found ways to change our protocols to improve the yield of our experiments Once the proper adjustments were made to the protocol we were able to assemble our clusters without any trouble What was the biggest achievement using this cloning method Our biggest achievement using this cloning method was that we were able to assemble a large number of fragments in a small amount of time We managed to resolve
67. nt cells with the reaction mixture Figure 19 An overview of the In Fusion Cloning Protocol Points of interests e Primer design and quality are critical for the success of the In Fusion reaction In Fusion allows you to join two or more fragments vector and insert or multiple fragments as long as they share 15 bases of homology at each end Therefore In Fusion PCR primers must be designed in such a way that they generate PCR products containing ends that are homolo gous to those of the vector In Fusion and Gibson Assembly resemble each other they both rely on the 3 gt 5 exo nuclease activity of an enzyme which produces single stranded ends Figure 20 The main difference between these methods is the fact that the repair ligation activity occurs in vivo while using In Fusion This characteristic has three direct consequences displayed in red in the disadvantages below 3 gt 5 exonuclease activity of poxvirus DNA polymerase 215 bp overlap 20 lt In Fusion enzyme chews back 3 gt 5 to produce single stranded ends IN VITRO Ld wv Repair and ligation IN VIVO joel e y O Qu c A eu ilo le Figure 20 In Fusion Joining of DNA 6E Disadvantages e Experiments and especially primer de sign must be scrupulous As this method is based on homologous Advantages Clone any insert into any location with any vecto
68. of undergrad and overgrad students can lead to nice products as we have tried to provide one for you in 2015 In order to compile a cloning guide several iGEM teams from all over the world have been con tacted to cooperate on this Finally fifiteen teams contributed in a fantastic way and a cloning guide consisting of the basics about nine different cloning methods and experiences of teams working with them has been realized Without the help of all collaborating teams this guide could never have been realized so we will thank all teams in advance This guide may be of great help when new iGEM teams edition 2016 and later are at the point of designing their project How to assemble the construct for you project is an important choice which is possibly somewhat easier to make after reading this guide We all the collaborating teams hope that you read this cloning guide with a lot of interest We wish you a lot of succes with the iGEM project and dont forget to have a lot of fun in the meantime NRP UEA iGEM iGEM TU Eindhoven iGEM Evry iGEM Bonn iGEM Minnesota iGEM Manchester Graz iGEM Sydney iGEM Vanderbilt iGEM York Carnegie Mellon iGEM iGEM Pasteur UIUC iGEM iGEM Stockholm iGEM Toulouse UCLA iGEM Kind regards TU Eindhoven Tort Gut dst o T In November 1973 a paper published by Stanley Cohen and Herb Boyer marked the start of bi otechnology The paper described a way to construct new functional bacterial plasmids
69. of our competition season We find 3A Assembly to be very user friendly with relatively few problems arising 24 What was the most dif ficult task The most difficult task was the planning stage When we realized that the two parts we wanted to assemble were both in chloramphenicol backbones we had to be flexible and look for alternate plasmids As iGEM moves more toward making sSB1C3 the standard back bone it actually becomes harder to mix and match the antibiotic resistance markers and design constructs for 3A Assembly Did the cloning method work as expected Yes we got results on our first try What was the biggest achievement using this cloning method We were able to assemble an IPTG sensor by combining a lac promoter with a ribosome binding site yellow chromoprotein terminator cassette Due to delays in other areas of our project we were unable to characterize our construct but the 3A Assembly went very smoothly What would be your tips and tricks if other teams are going to use this method If time allows we often give an extra hour or so for digestion and ligation in addition to what the protocol recommends UIUC iGEM Hi were team UIUC Illinois Our team consists of 10 undergraduates in addition to several advisors This year we focused on making the SCRIBE system designed by members of the Timothy Lu lab at the Massachusetts Institute of Technology MIT into a characterized Bio Brick compatible part Also
70. one experi enced preferentially Did the cloning method work as expected Yes What was the biggest achievement using this cloning method Cloning a small piece of RNA succesfully 00 5 z i O le E a Why did you choose this cloning method This method is well established in E coli and permits transformation with high efficiency which is a big advantage when cloning small inserts 42 Additional information e Clontech has made a short video in which they dive into the workflow of In Fusion Cloning Clontech has placed an advert in Nature Communications in which they discuss the efficiencies and possibilities of In Fusion HD Cloning iGEM Toulouse Our Team is composed of eleven students seven engineer ing students in fourth year of Biochemical engineering at INSA Toulouse and four students in Microbiology and Bio informatics at the University of Toulouse Paul Sabatier Furthermore eleven professors INSA UPS INRA CNRS advise us Diversity is a key aspect of our team due to the complementarity of the different formations offered by our University iGE Toulouse 2015 Ns x PPP id Tung x nad Ps v5 AA a TN i T Fis igure 22 Team aches of IGEM Toulouse Melany Tanchon Anthony David Thomas Exteberry Benoit Pons Marine Pons Alexandre Le Scornet Laetitia Chaumont Blandine Trouche Melissa David amp Louise Gody Protocols The protocols of team Toulouse describe varroa tests
71. orked well for us and we were able to implement it to assemble spider silk genes in a customizable modular fashion What was the most difficult task Performing ICA to assemble your gene is the most difficult and time consuming task Depending on the size of the final construct you may expect to tend to a reaction continu ously for three to four hours With practice a long construct may be assembled in as little as two hours On the other hand preparing and designing the pieces used in ICA and the workflow of pieces generated after ICA are fairly simple Did the cloning method work as expected No but we did get it to work ICA is sensitive to the specific sequence of the sticky ends on the monomers and may not work properly depending on the exact nucleotide sequence you wish to construct See the design protocol for more details What was the biggest achievement using this cloning method ICA was used successfully to construct our spider silk genes This validates the ability of ICA to work for modular repetitive sequences What would be your tips and tricks if other teams are going to use this method When planning to perform ICA set aside a block of about 3 4 hours In addition you will need a set of micropipets which you will be using for the entire time It is best if these pi pets are not shared with the rest of the team In addition it is helpful to prepare each exten sion reaction ahead of time so the total time spent doin
72. per discussing the advantages of differ ent assembly techniques in synthetic biology we suggest from the Imperial College of London titled Developments in the Tools and Methodologies of Synthetic Biology authors include Richard Kelwick James MacDonald Alexander Webb and Paul Freemont UIUC IGEM Why 3A Assembly Our team s project involves incorporating an analog biosensor one that can detect an input across a spectrum with genetic memory Part of our testing involved comparing the output of our device with traditional biosensor constructs consisting of a promoter plus a GFP reporter To assemble these constructs we plan to use a combination of traditional assembly and 3A assembly deciding on a case by case basis which is easier for the particular construct we have in mind We chose these methods because all of the parts required for our GFP constructs are already in BioBrick form making BioBrick specific cloning methods the natural choice Materials DNA Purification Kit We used Omega Bio Tek Restriction enzymes We used EcoRI HF Xbal Spel PstI from New England Biolabs NEBuffer such as CutSmart NEB 10X T4 DNA Ligase reaction buffer T4 DNA Ligase H O Destination plasmid as purified DNA Upstream and downstream parts as purified DNA Design considerations iGEM provides a very useful protocol in the help section of the Registry of Standard Biological Parts Our team follows this protocol but has also noted
73. prior been fragmented into smaller parts Advantages OE PCR allows side directed mutagenesis cloning of short fragments into specific vector positions OE PCR does not rely on restriction enzymes or ligation OE PCR is a very straight forward technique and easy to understand and perform OE PCR is highly efficient when a high fidelity HF polymerase and the right PCR condi tions are used HF Polymerases with 3 5 proofreading activity such as Pfu Phusion or Q5 are most recommendable Only few reagents are needed to perform OE PCR cloning such as HF polymerase dNTPs PCR Buffer flanked insert vector and ddH20 After performing OE PCR the insert will be found directly in the target vector and ready for characterization or further applications Disadvantages Using long inserts up to 100 nucleotides in length can lead to unspecific annealing and to false integration Necessity for restriction digestion not very reliable or sequencing PCR smears can arise from wrong PCR conditions too stringent primer fails to anneal or too relaxed non specific priming Hence PCR conditions can sometimes be a lot of trial and error Repetitive sequences can be troublesome for creating insert or for integrating into target vector Design conciderations 1 Design specific elongation primers 60 80 bp is optimal with which you can extract the insert sequence from genomic DNA or a plasmid backbone Adjust the primers that they ha
74. r of your choice Efficiently clone a broad range of frag ment sizes Clone multiple DNA fragments simulta neously into any vector within a sin recombination technology it does not work in any microorganisms probably due to different repair systems Cloning repair ligation occurs in vivo Therefore you cannot check the integri ty of your construction before transfor mation gle tube reaction No restriction digestion phosphatase treatment or ligation is required Final constructs are seamless with no extra or unwanted base pairs Possiblity to assemble large gene frag ments and generation of gene libraries AppacaNonS m Join multiple pieces of DNA within a single reaction see Figure 21 Insertion of point mutations see Figure 20 Delete and replace whole segments of DNA EEn 33 A Insert introns within cDNA Create modular expression vectors with interchangeable parts Swap domains on a gene Create seamless fusion proteins Figure 21 A Multiple and directional cloning B A DNA mutation strategy Frequently Asked Questions e What is the largest DNA fragment compatible with In Fusion Cloning DNA inserts up to 15 kb have been successfully cloned into pUC19 using In Fusion Cloning e What is the smallest DNA fragment compatible with In Fusion Cloning The smallest insert successfully cloned with In Fusion Cloning was a 50 bp oligonucleotide including two 15 nt homologous overlaps with the vecto
75. r se lected overnight during miniculture by recombination as a defense mechanism especially if you are cloning similar parts o Make sure that the genes or parts you are working with do not contain any restriction sites of the restriction enzyme you choose o Useofareporter gene enables checking if everything was inserted correctly To confirm succesful insertion a colony PCR can be performed Sequencing will give the final answer The iGEM Evry team Our team is composed of 8 undergraduate students from the University of Evry Val d Es sonne and the AgroParisTech engineering school This year we are engineering S cerevisiae to modulate the immune response by acting on the activity of dendritic cells Our main goal is to produce a synbio based immunotherapy presentation of tumoral antigens to the immune system and activation of it Figure 33 iGEM Evry team from left to right both rows Julie Zarowski advisor Cyrille Pauthenier advisor Francois Bucchini Pierre Yves Nogue Marjorie Aubert Cl ment De Obaldia Louise Barreau Fr d ric Ros Protocols This protocol describes the basic steps of Golden Gate cloning The link can be scanned in the following QR code IGEM Sydney Why Golden Gate Golden Gate was chosen as the primary cloning method over other prominent methods such as Gibson Assembly and Traditional Cloning as we were trying to clone more than one insert into the vector Even though Gibson Assembly is
76. r success rate may be due to lower levels of DNA damage induced by the blue light as opposed to UV light When cloning big protein coding inserts like ours it is best to keep their expression repressed when screening for the correct prod uct We also recommend doing all controls for the vector 5 end vector dephosphorylation reac tion and we recommend performing a gel extraction of the linearized vector if it contains a placeholder insert The control reaction is a self ligation reaction and subsequent trans formation To troubleshoot Gibson Assembly Reaction we did PCR controls of the reaction In some Cases we plan to use sequential Gibson Assembly where the final product is assembled in steps vector is added after several fragments have been joined the Gibson Assembly product can be itself used as a template for downstream applications iGEM York Our team is a group of undergraduate students that have an interest in synthetic biology and chose to investigate how to prevent eutrophication caused by downstream runoff from waste water treatment plants In particular we have spent the summer designing E coli to remove phosphate out of wastewater in bioreactors of wastewater treatment plants We are 10 under graduates 1st 2nd 3rd and 4th years that study either biology chemistry or biochemistry at the university We have had supervision from a few PhD students as well as several lecturers in the department UNIVERSITY of
77. r termini For In Fusion Cloning of short synthetic oligos between 50 and 150 bp the suggested oligo to vector molar ratio is 5 15 1 00 5 z i O le E a 5 e Can Iuse electroporation to transform the In Fusion Cloning reaction mix 1 ul of 1 5 diluted In Fusion Cloning reaction mix can be electroporated into 50 ul of electro competent bacterial cells 40 TM 9 e What bacterial strains are compatible with In Fusion Cloning o In Fusion Cloning requires bacterial cells with competency no less than 108 cfu ug supercoiled DNA The In Fusion Plus kits are supplied with Stellar Competent Cells so these cells may be a good starting point o Itis not recommended to use In Fusion on the following e TOP10 cells or their derivates e g ccdB Survival 2T1R E coli and related strains e g DH10B MC1061 are less optimal for In Fusion cloning resulting in a lower number of recombinant clones This may be of particular concern if you are performing multi ple fragment cloning or using a low copy vector E coli strains lacking recA1 or endA mutations E coli strains engineered for a particular application e 3 large scale protein expression Gram positive bacterial strains Bacterial cells carrying nupG deoR mutations IGEM Toulouse Why In Fusion Cloning This technology ensures easy single step directional cloning of any gene of interest into any vector at any locus In Fusion constructs are seamles
78. reduces the number of possibilities to about 3500 Therefore the order of the genes in each cluster can be changed and the order of the clusters inside the whole operon can be changed testing a number of possibilities Lox sequence 34bp Overlap Spacer Shine Dalgarno RBS LIR RIR Spacer Overlap Figure 16 Showing the design of the Lox sequence of IGEM Paris Pasteur iGEM Paris Pasteur Our team brings together fifteen bachelor and masters students trained in fields as varied as biology chemistry mathematics journalism and political science and coming for three major Universities in the Parisian region Pierre et Marie Curie University Paris Sud University and Sciences Po We are supervised by ten researchers post docs and PhD students within the Pasteur Institute specialized in various fields of research This year we decided to work on hindering the growing problem of plastic pollution To do so using E coli as a host we are designing a bacteria capable of degrading Plastic specifically PET and using the degradation products to synthesize Erythromycin A a commonly used broad spectrum antibiotic r ji An u gt d E l gt i d A M A A L1 bd n eni J Tum m m Lp 1 ADESSET RI L1 ug o NENS ee SERA d z gt EU 4 _ a 7 t NE ECT E e dI J S a 7 i E f x Leis lt ey 7 n e af og E ai i
79. roduce new errors the final assem bled product can be retrieved at high efficiencies Using this approach many of the costly and time consuming steps currently used to synthesize DNA including PCR and an error correction are eliminated SS P7 ARS PCR Gibson Assembly Mutated Plasmid Fisure 13 Multiple mutations can quickly be introduced to plasmids using Gibson Assembly Throush Site Directed Mutagenesis modified double stranded DNA fragments can be generated carrying the mutations within their overlaps Through Gibson Assembly these PCR products can be quickly assem bled into the final mutated plasmid This image was adapted from NEB Frequently Asked Questions How many fragments can be assembled within one Gibson Assembly reaction Different sources actually cite different numbers of fragments as the number of fragments depends on both the size as well as the sequence of the to be assembled fragments Gibson Assembly should in all cases however provide the correct clone if the number of frag ments within a single assembly does not exceed five If it is necessary to insert more frag ments it might be wise to do two sequential assemblies Does Gibson Assembly work with repetitive sequences Yes Gibson Assembly should work with repetitive sequences It is however important that the overlaps remain unique If the overlaps do resemble each other the correct DNA assembly may be produced at
80. rthermore it has a very straightforward methodology of cutting and pasting Lastly in terms of design and reagents it is one of the cheapest assembly methods Materials Restriction Enzymes Fermentas NEB Reaction Buffer often supplied with the enzymes gBlock IDT PCR product annealed oligos IDT digested DNA Plasmid backbone available from Addgene IDT NEB o Some of the more common ones are the pUC and pET series which are well documented and easy to use for beginners o Multiple cloning sites MCS variants can be used as a plasmid backbone To help with cloning in terms of finding restriction sites as well as visualizing the components and end product software is often helpful A Plasmid Editor ApE Genome Compiler gives free access to iGEM teams and has advanced features although it is difficult to use for beginners Design conciderations 1 Choose a vector appropriate for the project you are working on make sure the origin of rep lication is right for your model organism the antibiotic or auxotrophic marker is correct and that the appropriate tags promoters and terminators are in place Decide which enzyme s you wish to cut the vector and insert If you are using annealed oligos determine what overhang complements the vector s cut sites a Single enzyme sticky lack of specificity since the insert can attach itself in either orien tation b Single enzyme blunt lack of orien
81. rying the desired nucleotide changes Following the amplification with the mutated primers the fragments can be assembled into the final product e Assembly of large DNA constructs Worldwide laboratories are beginning to explore the use of synthetic biology in the pro duction of pharmaceuticals industrial compounds antibiotics cosmetics and alternative energy sources This often requires the assembly of a genetic pathway consisting of mul tiple enzymes and their associated regulatory elements Although template DNA is still required Gibson Assembly simplifies construction of the DNA coding for these types of molecules A long stretch of desirable DNA sequences can be broken down into several overlapping PCR products which can then be amplified by conventional PCR and com bined using Gibson Assembly e Assembly of chemically synthesized olignocleotides into dsDNA fragments Gibson Assembly can be used to directly assemble oligonucleotides into a cloning vector A common problem observed when chemically synthesizing long stretches of oligonucle otides is the introduction of errors To ensure that error free molecules are obtained at a reasonable efficiency a strategy employed by SGI and JVCI involves the assembly of only eight to twelve 60 base oligonucleotides with 30 bp overlaps at one time The resulting dsDNA is sequence verified and assembled into larger DNA fragments using the same approach Because assembly itself does not generally int
82. s coding sequences terminators etc Each part is flanked with a pair of convergent Bsal restriction sites Level 1 constructs are made by assembling level O modules to make a complete transcriptional unit The level 1 constructs can then be joined into level 2 or level M constructs with the use of a different type IIS restriction enzyme Bpil The process can then be repeated alternating between the two types of IIS restriction enzyme see Figure 29 3 For more information see e Weber 2011 e Engler 2014 Lo Ee z i Uu V lt e 42 fa Q ar o i le O JT Lf Frequently Asked Questions e How long does the entire process take To give a clear idea on the actual time needed when working with this cloning method take a look at the timeline in Figure 29 describing all the steps and their duration e How many fragments can be inserted and what is the expected yield It is generally admitted that you can clone up to ten fragments in one Golden Gate Assembly but we never tested it ourselves iGEM Evry The table on the right contains arbitrary values as they depend on our own experience Golden Gate Colony PCR Colony PCR Sequencing ABOUT 2 HOURS ABOUT 2 HOURS Q 1 5 HOURS results Two kinds of parts can z be used Miniculture Colony PCR e Subcloned parts e PCR Amplified parts e OVERNIGHT oe ABOUT 2 HOURS OR Best if more than 4 PCR Clean up fragments Q ABOUT 30 MIN Sequencing E col
83. s enabling translational reading frame continuity without any interfering scar sequences Design Conciderations 1 Every In Fusion primer must have two characteristics The 5 end of the primer must contain 15 bases that are homologous to 15 bases at the end of the DNA fragment to which it will be joined i e the vector or another insert The 3 end of the primer must contain a sequence that is specific to the target gene 2 The 3 portion of each primer should a betarget gene specific b be between 18 25 bases in length and have a GC content between 40 6096 c have a melting temperature T between 58 65 C You can use Oligocalc to work it out The Tm difference between the forward and reverse primers should be lt 4 C or you will not get a good amplification Note The T should be calculated based upon the 3 gene spe cific end of the primer and NOT the entire primer If the calculated Tm is too low increase the length of the gene specific portion of the primer until you reach a T of between 58 65 C d not contain identical runs of nucleotides The last five nucleotides at the 3 end of each primer should contain no more than two guanines G or cytosines C asno nol WIAD Ww 3 Avoid complementarity within each primer to prevent hairpin structures and between primer pairs to avoid primer dimers 4 Youcan perform a BLAST search to determine if the 3 end of each primer is unique and specific
84. sequentially totaling about 1 6 kb We have found this process to be fairly accurate with few single base pair mutations and virtually no incorrect monomer incorporation Literature indicates that ICA has been used to assemble 21 DNA pieces totaling about 1 9 kb e What are typical yields from ICA How reliable is it ICA itself yields DNA in sub nanogram amounts ICA in general is reliable but requires pre cision when setting up the reactions ICA is sensitive to mistakes in preparing the ligation reactions For example using the wrong cap in one reaction is enough to render the entire assembly unusable Useful resources The Predict a Secondary Structure server is an online tool which can predict the secondary structure of an arbitrary sequence The tool can be used to predict the hairpin of the cap UCLA IGEM Why Iterative Capped Assembly UCLA iGEM is working on creating and expressing silk from customizable spider silk genes which contain many repetitive sequences assembled sequentially We thought that ICA would be perfect for our project because it allows us a high level of control over the construc tion of our silk genes Materials e Streptavidin coated beads are essential for this method they act as the solid substrate for the elongating DNA chain We used M 270 streptavidin coated Dynabeads from Invitro gen A highly selective DNA ligase is preferred such as T7 DNA Ligase since correct assembly of the A Band
85. sert dsDNA fragment is used as a primer with corresponding flanking regions to the insertion site High Fidelity Polymerases show higher PCR efficiency High concentrations of insert and a relatively lower melting temperature 5 10 C under melting temperature can show higher efficiency The restriction enzyme Dpnl can be used to degrade the maternal template in order to in crease transformation efficiency 15 30 cycles can be used Studies show that at around 17 cycles the OE PCR efficiency is the highest Fragment elongation for each part you wish to combine aa U ar ci le E C OD Pur PS Ln Q x OD gt O JT Direct integration of elongated fragment into target vector lt Figure 43 One Fragment OE PCR for direct integration of a flanked insert into target vector OE PCR represents also a very powerful tool if you want to study a protein in an alternative version in a pre set expression vector You can insert or delete specific regions in the expression vector of your choice by designing specific primers with corresponding flanking regions In the example shown above you can insert any sequence whilst amplif ying the whole plasmid Applications This technique is a primary tool for site specific mutagenesis such altering nucleotides intro ducing deletions or insertions into specific sequence locations However it is also possible to clone with this technique longer genes into a plasmid which has
86. soseseseseoeoseseseseseesesesesssseseseseseo 64 AOL IVTBIBOSOO ace roti rii EEA EENAA ENEE OE miis dae 67 MIL GJ 9 2 A 9 8 9 OO S 69 MR gM Lem 72 12 Overlap Extension PCR wc cscssscsssscscsscscsssscsssscsssscsssscsssssscsssscssssssssssscsssssessscessscessscessscesesees 73 12 1 StOCKAOIMN woe RR 76 Traditional Cloning Introduction Experiments in the early 1950 s showed different growth behavior of bacteriophage X depend ing on which E coli strain was used as a host First the reason for this phenomenon was un clear However in the 1960s it was shown that this strain restriction is caused by an enzymatic cleavage of the phage DNA that in contrary to the bacterial DNA used a different methylation pattern of its genome and was thus recognized by certain endonucleases These first so called restriction enzymes were type I restriction endonucleases which means that they cleave the DNA not directly at its recognition site However in 1970 also type II restriction enzymes were found which cleave the DNA at its recognition site and therefore were way more suitable for scientific research The discovery and characterization of restriction enzymes not only marked the beginning of recombinant DNA technology but was also awarded with a Nobel Prize in 1978 F x EE CTCGAG 9 GAGCT C x 9 E amus E e H
87. t being said it seems that the number of fragments has a greater effect on transformation efficiency than the length of the inserts e How long does the homologous regions have to be The minimum amount of homology is 30 bp on both sides or 20 bp on one and 80 bpon the other 8 This being said we used 40 bp because we wanted to be safe e Can Iuse homologous recombination to add a nucleotide or drop a nucleotide in order to de stroy a restriction site in the homologous region It depends If the nucleotide is close to the insert 10 bp away then there should not be an issue as long as you still have a minimum of 30 bp of perfect homology If the nucleotide is in the middle or end of the homologous region you can extend the homology so that there is 30 base pairs of homology before the site e IfIPCR my inserts to increase their concentration do I need to clean up the PCR afterwards While we have not tested if this affects our transformation efficiency the efficiency has been high enough that we have concerned ourselves with testing this e Dolneedto purchase a separate miniprep kit for the yeast By using the lyse yeast solution we have been able to use the Promega and BioBasic bac terial miniprep kits to extract the plasmids from yeasts starting after the second step the lysis This being said we have noticed that our E coli transformations seem to have a lower efficiency that expected This is most likely because of the decreased
88. t difficult task was transforming the correct plasmid into the chassis We would iso late the correct plasmid but then transforming the plamid into the chassis required us to do a lot of troubleshooting and going through our protocol over and over making sure we did everything correctly Did the cloning method work as expected The cloning method did work as expected We did obtain the fluorescence for a few of the organisms we used We unfortunately could not obtain it for the PelB Gaussia due to the inconsistencies The inserts were excreted out of the inconsistend cells which made trans formation difficult Since transformation likelihood was low the fluorescence being seen was also very low What was the biggest achievement using this cloning method The biggest achievement was extracting and isolating the fluorescence from most of the organisms we used such as the firefly which we could use for our fluorimeter What would be your tips and tricks if other teams are going to use this method Some tips and tricks for other teams would be to make sure the plasmid is isolated correctly with few inconsistencies in the DNA sequence to make sure transformation is possible For a few of our plasmids since we had a few changed sequences the transformations failed sometimes which is why we had to redo some of it Design Conciderations Preparation of vector and insert Insert from a PCR product Design primers with appropriate restriction si
89. tation specificity and low ligation efficiency c Double enzyme sticky high specificity and ligation efficiency d Double enzyme blunt high specificity but low ligation efficiency Make sure the enzymes you are using have the proper locations and number of cut sites and that you are not using two isoschizomers same recognition site or isocaudomers same cleavage product Check the methylation sensitivities of the enzymes you want to use dam dcm CpG and ascertain if the plasmid you are cutting comes from a dam dcm or dam dcm strain Whenannealing oligos for your insert make sure to include the correct overhangs to corre spond to the sticky sites on the plasmid since digesting annealed oligos is inefficient due to their lack of overhang and low purify yield If you are ordering the DNA insert as a gBlock and the sequence has an improper restriction site use synonymous mutation to remove the recognition sequence NIq1opueA SI 00 E s f S G IA 16 Experiences How did you experience working with this cloning method This method is unique in the fact that there are so many variables to consider blunt end enzymes cut under different conditions than sticky end enzymes One has to keep track of the right digestion buffer to use for each enzyme as well as its period of effectiveness before star activity This can be done by looking whether an enzyme can be heat inactivated at w
90. tension PCR We wanted to create a novel chimeric receptor for which we wanted to integrate a molecule within the sequence of another protein After we failed to synthesize the chimeric protein via the IDT DNA Service we had to look for other possibilities and found OE PCR We needed a method which is not relying on restriction enzymes as we didn t want to create any scarring OE PCR seemed to be the appropriate tool to engineer quickly and easily our constructs We created within one week most of our planned chimeric constructs which shows that with a bit of preparation this technique is quite easy to apply and shows tremendous cloning efficiency Experiences e How did you experience working with this cloning method After we have been quite disappointed by the service of IDT in synthesizing our long gene fragments we were quite eager to see that this technique showed to be rather simple and we could construct our chimeric proteins in a functional plasmid backbone in a short time It is not easy to find the right PCR conditions for all inserts and overlap integrations but over time you will get a very good feeling at which temperature the melting temperature needs to be set and what the elongation time should be However you should be very care ful when you want to integrate repetitive sequences Those show that the yield of your PCR will reduce dramatically e What was the most difficult task The most difficult task was the design of prop
91. tes to clone unidirectionally into a vector Choose proofreading polymerases such as Phusion High Fidelity HF DNA Polymerase Use Phusion PCR to optimize plasmid and amplify DNA Purify the vector and insert using the PCR purification kit Then follow the QlAquick PCR purification Kit Protocol Using appropriate restriction enzymes cut the appropriate sites Cut out the samples in obtaining the DNA and repeat the PCR purification kit protocol in order to isolate the correct plasmid being analyzed iGEM Carnegie Mellon This is the Carnegie Mellon University iGEM team which consists of ten undergraduate stu dents There are five biologists and five engineers on the team ranging from majors of Elec trical amp Computer Engineering to Biology We work at Mellon Institute and do collaborations withUniversity of Pittsburgh Georgia and Eindhoven ERSITY Figure 7 Team CMU Left to Right Top Kenneth Li Jordan Tick Will Casazza Max Telmer Niteesh Sundaram Bottom Wei Mon Lu Dominique Cheylise Ruchi Asthana Donna Lee Michelle Yu Protocols e The protocols used were adapted from New England Biolabs Traditional Cloning Quick Guide References 1 Roberts RJ 2005 How restriction enzymes became the workhorses of molecular biology Proceed ings of the National Academy of Sciences of the United States of America 102 17 5905 8 2 https www embl de pepcore pepcore_services cloning cloning_methods restriction_enzy
92. the initiator but lacks biotinylation One end of the terminator is compatible to the reverse sticky end of the C type monomer unit This end is 5 phosphorylated to enable ligation The terminator also contains a primer binding site that can be used for PCR amplification as well as the bio brick suffix The capping oligos structures that are comprised of a single 5 phosphorylated ssDNA oligo that can form a stable stem loop structure with a unique sticky end There are three distinct caps each of which can bind to the A B or C sticky ends Qs A js Figure 26 Overview of the pieces which play a role in ICA A The 5 end of the top strand ACT G is biotinylated and the 5 end of the bottom strand TCAA is phosphorylated TCAA is the reverse com plement of the A sticky end The initiator contains the biobrick prefix start codon and 8X His Tag B The 5 end of the top strand of the terminator is phosphorylated The sticky end on the terminator A is AGGT which is the 5 sticky end C The capping oligos have a hairpin secondary structure The stem loop sequence is the same for all three caps The only difference is the sticky ends present The image shows the A sticky end Secondary structure prediction courtesy of the Predict a Secondary S Structure Web Server by the Mathews group D N Advantages Disadvantages e ICA is specifically designed for assembly e ICA is difficult it is less well suited tha
93. thod does not present any major difficulties Up to 4 fragments can easily be inserted in a single tube reaction with decent transformation yield The critical step is to design proper primers that fit each other What was the most difficult task The bottleneck of this method is the primer design as there are usually multiple fragments to clone Apart of this the Golden Gate method does not present any major difficulties in terms of bench work Did the cloning method work as expected Cloning worked as expected but screening is required after Indeed this method is not nec essarily as efficient as other cloning methods What was the biggest achievement using this cloning method The succes of the cloning itself What would be your tips and tricks if other team are going to use this method o Cloning several fragments using sub cloned parts rather than PCR amplified parts helps to get higher yields o Whendoing colony PCR the multiplex method is more reliable in order to check if all your parts were included in the assembly However it requires that you design a set of primers for each of your parts o Minipreps are cleaner than PCR clean up They produce a better yield and result in more reliable NanoDrop results If you still want to perform a PCR clean up you might prefer to use a gel quantifying method instead of NanoDrop as the yield is lower o Keepin mind that even if colony PCR results are encouraging parts can be counte
94. tion in a single step Golden Gate Cloning also involves the changing of antibiotics between parts and acceptors This allows for the selection of only the desired construct Steps like PCR and gel purification can be skipped 2 There are various assembly standards within Golden Gate Cloning which assign specific overhangs to different types of parts e g promoters coding sequences terminators so assem bly is no longer scarless but parts are standardized and interchangeable between labs Bsal Q gt oK N NNNNN n NNNNN N 52 Golden Gate Assembly Bsal amp T4 m DNA Ligase Digestion by Bsal yields the fragments g EN Ligation by T4 DNA ligase Figure 29 The one pot assembly of Golden Gate Cloning features ty pe IIS restriction enzymes which cut outside of the recognition sequence This yields user defined overhangs which can be used to assem be multiple parts in a pre determined order and orientation within a single pot reaction Different colors of overhangs correspond to different DNA Sequences The acceptor vector has a different antibiotic resistance to the parts allowing for the selective formation of the new product Note that the inserts and cloning vectors are designed to place the ty pe IIS restriction site distal to the cleavage site such that the ty pe IIS restriction enzyme removes the recognition site from the assembly The plasmid cannot be digested again after the Golden Gate Assembly
95. tne y d P Aa E ls E Be d a 7 e e ar e uL i s J ZEE 4 I A E 4 f e N E 1 T 4 e Ps m aa rj w D R d s Figure 17 Team photo of Paris Pasteur with Mathilde Ananos Valentin Bailly Jules Caput Alma Chapet Batlle Maxime Entremont Lucas Krauss Florence Moesch Thomas Neff K vin Plouchart Sertac Tas Am lie Vandenda le Pierre Vilela IGEM York Why Gibson Assembly We chose Gibson Assembly because we wanted to benefit from IDT s DNA synthesis offer and we considered this technique the optimal method in terms of speed and efficiency The use of synthesized oligos requires only small DNA amounts and hence prior cloning steps are not necessary Materials For the design of our experiments we used the following materials pSB1C3 vector from the Registry of Standard Biological Parts gBlocksQ offered by Integrated DNA Technologies Primers by Integrated DNA Technologies up to 6Obp The NEBuilder Cloning Kit supplied by New England Biolabs SnapGene The Q5 High Fidelity Polymerase Master Mix offered by New England Biolabs Phusion High Fidelity DNA Polymerase by by New England Biolabs GoTaq G2 by Promega for colony PCR screens Zymoclean TM Gel DNA Recovery Kit by Zymo Research DarkReader Blue Light Transilluminator SYBR Safe SureVector MasterMix by Agilent Technologies Inc a polymerase cycling assem
96. to a double stranded piece of DNA Good design beforehand also allows the inclusion of overhang that would create sticky ends compatible with the oth er digested sequences This allows for the inclusion of short sequences lt 80 bp into a plasmid without ordering expensive dsDNA and gBlocks or trying to isolate small sequences from other plasmids or PCR products which is difficult when the DNA is less than 100 bp e Multiplex Assembly Using traditional cloning techniques there is the possibility of assembling several inserts at once into a backbone Although other techniques like biobrick assembly allow simultaneous inclusion of two inserts and Gibson assembly can put together more than two but requires design of large homologous regions traditional cloning has been known to construct plas mids from three or more inserts The unique sticky ends that can be constructed on the 5 and 3 end of each DNA sequence and the vector permits a theoretically unlimited number of fragments that can be ligated together due to the fact that there is only one unique ori entation in which they will form a complete plasmid This is a convenient way of putting together several sequences at once especially when coupled with an RFC protocol that ex pands the number of isocaudomers ame P t Q Qu c A P j o le Additional information DoubleDigest calculator by Thermo Scientific Thermo Scientific If one is using two different restri
97. to produce a scar that can no longer be cut C6 Cut PCR Product Cut with E amp S Lo with X amp P LUCES EX S X S P m C Mix amp Ligate E X M S P Figure 8 A diagram showing the digestion scheme used for 3A assembly When the two parts you wish to combine A and B in the figure are digested with the appropriate enzymes they can be ligated to create a scar that is no longer recognizable by the enzymes When cells containing the construct are plated on the same antibiotic which the backbone C in the figure confers resistance to most of the sur viving colonies will contain the parts cloned into the vector 3 This image was adapted from iGEM 3A Assembly 20 Points of interests The parts being used must have a BioBrick prefix and suffix The part inserts must have different antibiotic resistance from the backbone otherwise there is no way of selecting for the correct construct It is sometimes possible for a parent part plasmid to be transformed into the clone along with the desired construct If you suspect this has happened it s advisable to screen the clone for resistance to the parent s antibiotic 5 Figure 9 Culture plates showing the process of 3A assembly Parts are seperated from colonies with a different antibiotic resistance marker than the marker present in the destination vector enabling insertion of two fragments within a single reaction bd 5 re y le Qu c A f
98. ttempt As noted by the developers larger fragments are harder to assemble We have successfully cloned frag ments of total size of 450 bp 1116 bp 2100 bp and We have also assembled large constructs with long PCR products 3600 bp and 4800 bp The PCR products are derived from Colony PCR reactions with a high fidelity polymerase The Colony PCR reaction we used was a touch up colony PCR with primers that contain overhangs Since the initial annealing is partial the annealing temperature has to be lower than the annealing temperature of the whole primer with the overhang We also managed to perform site directed mutagenesis simultaneously with our cloning and assembly What was the biggest achievement using this cloning method The biggest achievement has been cloning a 4 8 kb Phosphate specific ABC transporter from Sinorhizobium meliloti as one big Colony PCR product It was difficult to purify the PCR product as large DNA fragments are tightly bound to the silica membrane of the puri fication column we were using Extra elution steps at high temperature were required What would be your tips and tricks if other teams are going to use this method We would recommend gel extraction using a Zymo Gel Extraction kit or similar which has a smaller elution volume gt 6 uL and a smaller membrane delivering more concentrated eluates Also we got really good results using SYBR Safe and Dark Reader Blue Light Tran silluminators We think our highe
99. ve approximately the same melting temperature The flanking regions 25 27 bp to the insert sequence need to be homologous to the sequence of the target vector Simulate the PCR reaction in Snapgene to confirm the feasibility of your OE PCR Perform a high fidelity PCR by using for example Q5 High Fidelity Polymerase Master Mix and purify the PCR product by first digestion with DpnI and the purification of the desired PCR product either by gel electrophoresis or using the PCR purification Kit from QIAGEN The purified flanked insert fragment can now be used as primers for whole plasmid ampli fication of the target vector In this PCR you should again use a high fidelity polymerase as they achieve better results in cloning efficiency and how mutations rates Recommended The parental plasmid can be digested with DpnI to reduce the background Recommended You can also first linearize the target plasmid with the corresponding flanking regions at the end This may be especially useful while having long target plas mids gt 5 kbp Purify the plasmid after DpnI digestion using the PCR purification Kit from Qiagen This can then be used for transformation Yields of more than gt 60 are normally to expect but dependent highly on the construct sequence flanking regions and tertiary structure Recommended Colonies should be sequenced to exclude the possibility of point mutations uononponu IGEM Stockholm Why Overlap Ex
100. you experience working with this cloning method It was very interesting working with our cloning method in part because our construct contained a bidirectional promoter At first it was frustrating because our first two trans formations failed however once we realized that we had switched our linearized plasmids we had great and good smelling success e What was the most difficult task Besides dealing with human error the most difficult task was designing the gBlocks to eliminate illegal restriction sites Using tools such as ApE or Snapgene to create the final sequence that you want can greatly help to visualize what you are doing eyoseuur IO e Did the cloning method work as expected Yes the yeast recombination seems to have worked well L9 e What was your biggest achievement with the cloning method We were able to clone 2 fragments totaling 3 3 kb of non homologous region into a yeast plasmid e What would be your tips and tricks if other teams are going to use yeast recombination o Watch for excessive homology within your inserts as the yeast might perform homolo gous recombination on that area as well o Once you have successfully transformed the yeast make sure you keep it on the selec tive media otherwise it will eventually kick out the plasmid o Ifyou don t have access to or a budget for purchasing a yeast plasmid you can make any plasmid a yeast vector by adding a yeast origin of replication and selection marker T

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