BacLoxP Baculovirus Expression System
Contents
1. ATG biosynthetics de a gt a ax PEE Solutions E in DIOGENESYS a o gt Toggle Expression System Series flEXLoli Multi Protein Expression in E coli User Manual Version 1 1 January 15 2014 TABLE OF CONTENTS A ALEXcColi kit contents Reagents to be supplied by user see also section D Protocols B Escherichia coli a premier laboratory workhorse B 1 Introduction C A Novel Approach to Multigene Applications C 1 The Toggle Concept a Primer C 2 Transfer vectors Acceptor Donor recombineering system C 3 Generating multi gene expression cassettes C 3 1 Creating individual gene expression cassettes C3 1 1 Standard Approach C3 1 2 Inserts with Other Unique Restriction Sites C 3 2 Multi gene construction via Toggle multiplication C 3 3 Multi gene construction using Cre Lox recombination C 4 Introducing additional control elements D Protocols D O Introductory remarks D 1 Cloning into TogColi transfer vectors D 2 Conversion of donors into conditional donors D 2 1 Protocol 1 Conversion into conditional oris D 3 Concatenation of genes gene cassettes by Toggling D 3 1 Protocol 2 Adding gene cassettes through toggling D 4 Deconstruction of gene cassette assemblies D 4 1 Protocol 3 Disassembling multi gene construct to regenerate individual expression boxes D 5 Cre LoxP reaction of Acceptors and Donors D 5 1 Protocol 4 Cre LoxP fusion of Acceptors and Donors D 5 2 Prot2. 11 12 13 14 15 16 17 18 19 45 References Alberts B 1998 The cell as a collection of protein machines preparing the next generation of molecular biologists Cell 92 291 294 Ausubel F Brent R et al eds 1994 Current Protocols in Molecular Biology John Wiley amp Sons New York electronic version DOI 10 1002 0471142727 Bhattacharya A 2009 Protein structure Structures of desire Nature 459 24 27 Charbonnier S Gallego O Gavin AC 2008 The social network of a cell Recent advances in interactome mapping Biotechnology Annual Review 14 1 28 Chemler JA and Koffas MAG 2008 Metabolic engineering for plant natural product biosynthesis in microbes Current Opinion in Biotechnology 19 597 605 Chou CP 2007 Engineering cell physiology to enhance recombinant protein production in Escherichia coli Applied Microbiology and Biotechnology 76 521 532 Estrozi LF Boehringer D Shan SO Ban N Schaffitzel C 2011 Cryo EM structure of the E coli translating ribosome in complex with SRP and its receptor Nature Structural and Molecular Biology 18 88 90 Gaiser F Tan S and Richmond TJ 2000 Novel dimerization fold of RAP30 RAP74 in human TFIIF at 1 7 resolution Journal of Molecular Biology 302 1119 1127 Hunt 2005 From gene to protein a review of new and enabling technologies for multi parallel protein expression Protein Expression amp Purification 40 1 22 Ino
3. ATAACTTCGTATA GCATACAT TATACGAAGTTAT 3 3 TATTGAAGCATAT CGTATGTA ATATGCTTCAATA 5 inverted repeat Spacer inverted repeat Figure 13 LoxP imperfect inverted repeat In a reaction where several DNA molecules such as donors and acceptors are incubated with Cre recombinase the fusion excision activity of the enzyme will result in an equilibrium state where single vectors educt vectors and all possible fusions coexist Donor vectors can be used with acceptors and or donors and vice versa Higher order fusions are also generated where more than two vectors are fused This is shown schematically in Illustration 14 The fact that Donors contain a conditional origin of replication R6Ky that depends on a pir oir positive background now allows for selecting out from this reaction mix all desired Acceptor Donor s combinations For this the reaction mix is used to transform pir negative strains TOP10 DH5a HB101 or other common laboratory cloning strains Then Donor vectors will act as suicide vectors when plated out on agar containing the antibiotic corresponding to the Donor encoded resistance marker unless fused with an Acceptor By using agar with the appropriate combinations of antibiotics all desired Acceptor Donor fusions can be selected for FIEXiColi System ATG biosynthetics GmbH 21 OOd SINGLE Gm ColEl Kn R6Ky clk R6Ky Acceptor Donor1 Donor 2 AD Acc Don1 Acc Don2 TRIPLE ADD 9197 UO
4. files and accompanying files from ATG at order atg biosynthetics de In the next section the acceptor and donor vectors are presented as circle maps and in addition the multiple cloning site MCS of each vector is shown featuring important unique restriction sites Moreover you will find for the purposes of designing a restriction strategy a non exhaustive list of restriction endonucleases that cut once twice or not at all Additional restriction sites can be identified with any sequence analysis software e g VectorNTI ApE etc or by using online tools such as WebCutter 2 0 http rna lundberg gu se cutter2 or the NEB cutter V2 0 http tools neb com NEBcutter2 Finally for verifying vector identity representative digestion patterns are listed in a table FIEXiColi System ATG biosynthetics GmbH 40 E 3 1 Acceptor vectors E 3 1 1 TogColiAccA1 5 amp A1 6 2572 bp 2623 bp LoxP T7 Promoter lac Promoter DS Insertion Site Trosition Site T7 ColE1 ColE1 Terminator ori T7 ori Terminator flEXColi Acc A1 6 GmR 2623 bp J ALEXCColi yin Acc A1 5 GmR 2572 bp Split ori GentR GentR TogColi A1 5 GmR TogColi A1 6 GmR Features T7 and lacT promoter respectively T7 polyA signal terminator loxP recombination signal sequence Gentamycin resistance marker Split ColE1 R6Ky origin of replication Sequence Please request from order atg biosynthetics d FIEXiColi System
5. Absl Asc GAS Box BssHIl V2 BssHil MauBl BssHll SgrDI AsiSl R Figure 11 Schematic of gene cassette concatenation by toggling via the inner pair of restriction sites The strategy and de facto restriction enzymes are detailed in the text The resulting two gene assembly is immune to disassembly with Ascl MauBl as indicated by the no sign in step 4 but the individual gene cassettes can be retrieved for re use by BssHII restriction as described later see fig 18 By the same logic more gene cassettes can be added and in theory a mere two selectors are required see figure 12 These are used alternatingly thus you toggle back and forth between the selectors FIEXiColi System ATG biosynthetics GmbH 19 Figure 12 Schematic drawing of the process for building a three gene assembly by toggling between constructs with two selectors red Absl Ascl g and green solid arrow By the same logic constructs with more gene cassettes can be pieced together but for practical purposes assemblies with more than four genes are Absl Ascl Asi 3 usually not recommended Y Abs Ascl_ AsiSI The no sign red circle slash indicates that the hybrid ligation juncture cannot be restricted ira MauBl with either of the restriction enzymes used in the initial cloning procedure The resulting fragment is placed into the multiplication module of another acceptor or donor vector containing one or multipl
6. ATG biosynthetics GmbH 41 E 3 2 Donor vectors E 3 2 1 TogColiDonA1 5 2774 bp KnR 2619 bp CmR LOXP 57 Promoter 9P T7Promoter CDS Insertion Site CDS Insertion Site T7 Terminator T7 Terminator ColE1 ori ALEXCCO i PLEX Oli Split Split Don A1 5 KnR Le Don A1 5 CIR ori 2584 bp 2462 bp ChIR KanR TogColi A1 5 KanR TogColi A1 5 ChIR Features T7 promoter T7 polyA signal terminator loxP recombination signal sequence Kanamycin chloroamphenicol resistance marker Split ColE1 R6Ky origin of replication Sequence Please request from order atg biosynthetics de FIEXiColi System ATG biosynthetics GmbH 42 E 3 2 2 TogColiDonA1 6 2864 bp KnR 2693 bp CmR LOXP tac Promoter LoxP lac Promoter _ CDS Insertion Site CDS Insertion Site T7 Terminator T7 Terminator ColE1 ori AUEXCCOl Ppa Don A1 6 cIR 2513 bp AEXCOII Don A1 6 KnrR 2635 bp Split ori KanR TogColi A1 6 KanR TogColi A1 6 ChIR Features lacZ promoter T7 polyA signal terminator loxP recombination signal sequence Kanamycin chloroamphenicol resistance marker Split ColE1 R6Ky origin of replication Sequence Please request from order atg biosynthetics de FIEXiColi System ATG biosynthetics GmbH 43 E 3 3 Analytical restriction digest patterns All FlexiColi acceptor and donor vectors yield characteristic restriction digest patterns when plasmid DNA is restricted with Hindlll and or Hinfl or a
7. MauBl and Ascl SgrDI produce polar overhangs as indicated by the arrows that can be used for directional cloning FIEXiColi System ATG biosynthetics GmbH 29 2 digesting with BssHIl fig 17C and 18 to release all individual expression cassettes from the entire multigene assembly Such a complete disassembly is possible if the external restriction sites have not excluded from toggling The BssHII digest will give you a mixture of all individual cassettes The individual expression cassettes possess identical ends and thus are not polar in terms of the overhangs created by endonuclease restriction Subsequent re insertion into any design will therefore require either diagnostic digest or sequencing to ascertain a desired orientation Absl Ascl MauBl SgrDI AsiSl Y BssHll BssHIl BssHIll BssHIl BssHIl BssHiIl Figure 18 Releasing individual gene cassettes from multi gene assemblies via BssHII restriction While you cannot cut the hybrid restriction sites no sign with either Ascl or MauBl you can release the individual gene cassettes by restricting with BssHIl which is the 6 mer core sequence of both Ascl and MauBl FIEXiColi System ATG biosynthetics GmbH 30 D 5 Cre LoxP reaction of Acceptors and Donors D 5 1 Protocol 4 Cre LoxP fusion of Acceptors and Donors This protocol is designed for generating multigene fusions from Donors and Acceptors by way of the Cre LoxP reaction Reagents Cre recombinase f
8. on the phage lambda pL and or pR promoters Microbial Cell Factories 9 18 Van Hijum SA Medema MH and Kuipers OP 2009 Mechanisms and evolution of control logic in prokaryotic transcriptional regulation Microbiology and Molecular Biology Reviews 73 481 509 FIEXiColi System ATG biosynthetics GmbH 46 NOTES FIEXiColi System ATG biosynthetics GmbH 47 G Purchaser Notification Limited Use Label License No EX FC 1 ATG biosynthetics technology The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include bu
9. vectors have been constructively optimized to maintain a constant distance the promoter and the translation start codon indicated by the distance between R3 Ndel in fig 15 Additional control elements can be inserted upstream of the promoter e g between Absl and Ascl or Absl Ascl and Ndel which would correspond to the purple arrow in fig 15 FIEXiColi System ATG biosynthetics GmbH 23 D Protocols D O Introductory remarks Please note that the bacteria in the agar stabs have not been made competent for transformation If you wish to use them to transform your constructs you will have to prepare competent cells from the agar stabs in the kit if included in your configuration This applies specifically to the pirHC and pirLC strains used to maintain and amplify donor constructs You may follow your preferred protocol for preparing chemically or electrocompetent cells e g Inoue et al 1990 or variations of this protocol or standard protocols as described in Current Protocols in Molecular Biology or Sambrook and Russell Molecular Cloning 3 edition 2001 or older versions D 1 Cloning into TogColi transfer vectors Reagents Restriction endonucleases Ndel Bcll caution restricts only DNA from dam E coli strains DNA modifying enzymes DNA Phosphatase DNA ligase F coli competent cells Antibiotics Chloramphenicol Gentamycin Kanamycin The genes of choice can be either provided as a fully synthetic construc
10. within pairs are fully compatible with one another The inner pair can be used for ceicececa deconstructing Toggle assemblies whereas the outer pair can be used to build higher order multi gene constructs The spacer between Absl Ascl and MauBl SgrDl respectively can be used to integrate additional functions for recombination cloning etc Pal TogTec g SgrDI CGIICGACG T Box S Box This manual presents a new system with a set of novel transfer vectors specifically designed for synthetic biology applications but flexible enough to accommodate standard protocols The design of the vectors adheres to a stringent Toggle vector design see fig 2 and 5 that is common to all FIEXiColi System ATG biosynthetics GmbH cloning expression vectors of this series Toggle vectors combine established time tested elements with a new approach to the construction and expression of multigene plasmid constructs FIEXiColi System ATG biosynthetics GmbH 10 C 2 Transfer vectors Acceptor Donor recombineering system The Acceptor vectors TogColiA1 5GmR and TogColiA1 6GmR host time tested T7 and lac promoters and T7 polyA signal sequences see fig 6 A multiplication module M defined by the Ascl and MauBl see figure 6 allows integration of multiple gene cassettes ORFs and associated regulatory regions as schematized in fig 4 as TogTec vectors do not contain a classical multiple cloning site see appendix in their A boxes alt
11. LIS IIOLQNIUY Acc Don1 Don2 Figure 14 Cre and De Cre reaction cascade Schematic representation of Cre mediated assembly and disassembly of one acceptor with one or two donors respectively LoxP sites are shown as red circles resistance markers and origins are labelled White arrows stand for the entire expression cassette including promoter terminator and gene integration multiplication elements in the TogTec vectors Not all possible fusion products are shown for reasons of clarity Levels of multiresistance are indicated right side AD acceptor donor fusion ADD acceptor donor donor fusion FIEXiColi System ATG biosynthetics GmbH 22 C 4 Introducing additional control elements Protein expression requires certain genetic elements at the DNA and RNA level for it to work efficiently as exemplified by the Shine Dalgarno sequence in bacterial mRNAs Without this ribosome binding motif efficient translation of the corresponding mRNAs is compromised Yet many genes mRNAs lack such a consensus sequence motif which is not an absolute must for successful translation CODING SEQUENCE nt variable Figure 15 Constructive layout of expression boxes for use with the flexiColi system Ndel and Bcll define the coding sequence R4 defines a restriction site that can be introduced to exchange variants in the leader region that can extend from approx 40 to 40 nts relative to the translation start start codon The ToggleTec
12. a steady state or a developing differentiating cell this becomes more difficult for complexes that are transient in nature appear only periodically in cells or simply occur only in low amounts In such cases systems come in handy that allow homo or heterologous expression of these complexes in large amounts Such a large scale protein production approach by using recombinant technologies makes analysis of the structure function relationships in multi protein complexes more likely While various methods and systems have been developed to address this problem most of them are of little use for intense research efforts directed at generating and investigating scores of protein complexes in parallel i e in an automated fashion Such a system should be robust and easy to install in terms of manipulation operations and protocols and or components used in the process Hunt 2005 In addition they should provide enough flexibility to rapidly alter the multiprotein components which is a prerequisite for revising expression studies The ALEXColi system addresses these needs and avoids some problems that occur with in vitro reconstitution of protein complexes from individually or co expressed protein components FIEXiColi System ATG biosynthetics GmbH Protein Structure Protein Determination Bacterial Production Display Vector Development Metabolic nn R amp D Industrial Engineering Biotech Pathway Strain Design Develop
13. and combine the resulting fragments with a PCR amplified resistance marker by recombination SLIC or blunt end ligation using 5 phosphorylated primers Note that resistance markers can also be exchanged in between ACEXcColi vectors by restriction digestion with SgrDI and AsiSI C 3 1 Creating individual gene expression cassettes C3 1 1 Standard Approach The acceptor and donor vectors are suited for generating multi gene expression constructs from individual gene expression cassettes complete with regulatory regions such as promoter and terminator via Toggle multiplication All flexiColi vectors contain identical A box flanking rare cutter restriction sites One initial basic design condition is that none of the following restriction sites that are virtually never used for standard cloning strategies may be present in the gene cassette Ascl Absl MauBl SgrDI Pacl AsiSl Ndel Bcll ApaLl As a logical consequence Xhol BssHII Sall and Pvul are off the list as well as these constitute core sequences of the 8 cutters ATG offers a suitable gene design service that considers all these requirements The Toggle system is designed for integrating synthetic genes but can also accommodate other sequences as long as they adhere to certain design principles to fully exploit the advantages of the system Avoid Ascl Absl MauBl SgrDI Pacl AsiSl Pvul Ndel Bcll ApaLl Xhol BssHII Sall in your gene design ATG performs all t
14. are subject to multiple limited use label licenses the terms of the most restrictive limited use label license shall control ATG will not assert a claim against the buyer of infringement of patents owned or controlled by ATG which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser does not agree with the terms or limitations of this limited use agreement the customer can return the product at their own costs but for a full refund excluding shipping tax and handling fees For information about purchasing a license to use this product or the technology embedded in it for any use other than for research use please contact Out Licensing ATG biosynthetics GmbH D 79249 Merzhausen Weberstr 40 Germany or outlicensing atg biosynthetics com Note However in accepting ATG s material transfer limited label use agreement buyers are encouraged to disseminate the material to their colleagues if the recipient is willing to sign this agreement prior to transfer of the material combined with its regular purchase from ATG TOGTEC vectors are for research purposes only By signing the order form and on receipt of the TOGTEC vectors you agree not to distribute TOGTEC ve
15. assembly Correct integration is selected by virtue of the introduced selector D 3 1 Protocol 2 Adding gene cassettes through toggling Reagents required Restriction enzymes Ascl Xhol MauBl BssHIl AsiSI Pvul and enzyme buffers DNA phosphatase of choice T4 DNA ligase and buffer F coli competent cells Antibiotics Step 1 Prepare vector backbone and insert s Restriction reactions are carried out in 25 ul reaction volumes but can be scaled to any desired volume Enzymes are used according to the manufacturer s recommendations Accepting plasmid gt 1 ug in ddH O buffer x ul N x restriction enzyme buffer 25 N ul 10 mM BSA if required 2 5 ul MauBl BssHII 1 ul ca 10 U AsiSl Pvul 1 ul ca 10 U ddH O ad 25 ul FIEXiColi System ATG biosynthetics GmbH 26 Digest at 37 C or any other recommended temperature for at least 1 to 2 hours If your design does not allow using the six cutters Sall and or Pvul use the eight cutters SgrDI and AsiSl instead after checking your sequences for these restriction sites Donating plasmid gt 1 ug in ddH O buffer x ul N x restriction enzyme buffer 25 N ul 10 mM BSA if required 2 5 ul Absl Xhol 1 ul ca 10 U AsiSI Pvul 1 ul ca 10 U ddH O ad 25 ul You can separate the vector backbone receiving plasmid and the insert donating plasmid on an agarose gel excise the desired bands and extract the DNA using commercial kits Qiagen MachereyNagel etc or any alte
16. be directly mixed with 100 ul electrocompetent cells and transformed by using an electroporator e g BIORAD E coli Pulser at 1 8 2 0 kV Larger volume of De Cre reaction must be desalted by ethanol precipitation or PCR purification column prior to electrotransformation The desalted De Cre reaction mix should not exceed 0 1 volumes of the electrocompetent cell suspension The cell DNA mixture can be immediately used for electrotransformation without prior incubation on ice 5 Add up to 400 ul of LB media or SOC media per 100 ul of cell DNA suspension immediately after the transformation heat shock or electroporation 6 Incubate the suspension in a 37 C shaking incubator recovery For recovery of partially deconstructed double triple fusions incubate the suspension in a 37 C shaking incubator for 1 to 2 hours For recovery of individual educts incubate the suspension in a 37 C shaking incubator overnight or for at least 4 hours 7 Plate out the recovered cell suspension on agar containing the desired combination of antibiotic s Incubate at 37 C overnight 8 Colonies after overnight incubation can be verified directly by restriction digestion at this Stage refer to steps 12 16 This is especially recommended in cases where only a single educt or partially deconstructed multifusion plasmid is desired For further selection by single antibiotic challenge on a 96 well microtiter plate continue with step 9 Seve
17. col D 2 1 Protocol 1 Conversion into conditional oris Cleave the donor vector with ApaLl Dephosphorylate the cleaved vector Test successful dephosphorylation via transformation no colonies should appear Repeat until no empty vector religation product remains Perform ligation following standard procedures L 2 3 4 5 Cleave the donor with Sapl 6 7 Transform re ligated vector into pir E coli strains 8 Plate and select on LB agar plates contaning kanamycin or chloroamphenicol D 3 Concatenation of genes gene cassettes by Toggling As indicated in figure 11 page 18 gene cassettes can be added to AEX Coli vectors by replacing the S box in the receiving plasmid with a gene cassettes plus selector A and S boxes from a donating plasmid In principle two antibiotic resistance markers will be sufficient to alternate between selectors while building multigene assemblies and as this occurs in an alternating fashion the process is called toggling FIEXiColi System ATG biosynthetics GmbH 25 R3 T pCDS pCDS I Y i RO R4 T V pCDS ll pCDS lll y Y A O m RO R5 T pCDS pCDS I pCDS II pCDS IV Y y E A Figure 16 Adding gene cassettes via toggling Cycle 5 V One gene cassette is added with every restriction ligation cycle In its most basic application toggling can be accomplished with only two selectors here red and green that are alternately introduced into the growing multi gene cassette
18. combination of both Vector Length bp Restriction Fragment s bp TogColiA1 5GmR 2572 Hinfl 1177 883 267 245 Hinfl Pacl 883 616 561 267 245 TogColiA1 6GmR Hinfl Pacl 1179 616 561 267 TogColiA1 5KnR Earl Sapl 1135 36 734 33 505 210 TogColiA1 6KnR Earl Sapl 1186 87 734 33 505 210 TogColiA1 5CmR Hinfl 965 831 666 TogColiA1 6CmR Hinfl 1682 831 Hinfl SnaBl 1137 831 545 E 3 4 Sequencing primers To analyze integrity of your inserted genes of interest you may either use internal primers to sequence inside out or use the following primers to sequence outside in Please note that these primers have originally been designed for quality control of the native transfer vectors which is why there is only one for acceptors and donors respectively Toggle Universal Sequencing Primers TUP and TUSPs for sequencing inserts TUP 1 fwd 29nt 5 CAA CAG GTT GAA CTG CTG ATC TTC AGA TC 3 Binds 5 of Absl and should identify the A box from the 5 end Sequencing Primers to verify sequence identity of vectors if required TUSP 1R rev 19nt 5 CAC TTA ACG GCT GAC ATG G 3 Binds 3 of the antibiotic resistance marker TUSP 1F fwd 20nt 5 CGA TAG TTA CCG GAT AAG GC 3 Binds centrally in the split ori TUSP 2R rev 19nt 5 CAC GCT GTA GGT ATC TCA G 3 Binds centrally in the split ori FIEXiColi System ATG biosynthetics GmbH 44 FIEXiColi System ATG biosynthetics GmbH F 1 10
19. ctors yourself and that you will only use the vectors in your individual lab environment For all other purposes a specific and separate license needs to be acquired Third parties who come into possession of the TOGTEC vectors through the purchaser s distributing them need to sign the same agreement formally order the vectors at ATG and reimburse ATG for permission to use them FIEXiColi System ATG biosynthetics GmbH
20. ctrotransformation without prolonged incubation on ice 5 Add up to 400 ul of LB or SOC medium per 100 ul of cell DNA suspension immediately after the transformation heat shock or electroporation FIEXiColi System ATG biosynthetics GmbH 31 6 Incubate the suspension in a 37 C shaking incubator overnight but for at least 4 hours recovery period To recover multifusion plasmid containing more than 2 resistance markers it is strongly recommended to incubate the suspension at 37 C overnight 7 Plate out the recovered cell suspension on agar containing the desired combination of antibiotics Incubate at 37 C overnight 8 Clones from colonies present after overnight incubation can be verified by restriction digestion at this stage refer to steps 12 16 This quality control step should be carried out especially when only one specific multifusion plasmid is desired For further selection by single antibiotic challenges on a 96 well microtiter plate continue with step 9 Several to many different multifusion plasmid combinations can be processed and selected in parallel on one 96 well microtiter plate 9 For 96 well antibiotic tests inoculate four colonies from each agar plate with different antibiotic combinations into approx 500 ul LB medium without antibiotics Incubate the cell cultures in a 37 C shaking incubator for 1 2 hours 10 While incubating the colonies fill a 96 well microtiter plate wit
21. e gene cassettes The restriction sites involved in the initial cloning process step 1 and 2 in fig 11 are eliminated in the process as they become hybrid sites that can be cleaved by neither enzyme and multiplication can be repeated iteratively using the module present in the inserted cassette Nonetheless the individual gene cassettes can be re cycled by digesting the entire assembly with e g BssHII see fig 18 FIEXiColi System ATG biosynthetics GmbH 20 C 3 3 Multi gene construction using Cre Lox recombination Cre recombinase is a member of the integrase family Type topoisomerase from bacteriophage P1 It recombines a 34 bp loxP site in the absence of accessory protein or auxiliary DNA sequence The loxP site is comprised of two 13 bp recombinase binding elements arranged as inverted repeats which flank an 8 bp central region where cleavage and the ligation reaction occur The site specific recombination mediated by Cre recombinase involves the formation of a Holliday junction HJ The recombination events catalyzed by Cre recombinase depend on the location and relative orientation of the loxP sites Two DNA molecules for example an acceptor and a donor plasmid containing single loxP sites will be fused translocation The Cre recombination is an equilibrium reaction with 20 30 efficiency in recombination This provides useful options for multi gene combinations for multi protein complex expression 13bp 8bp 13bp 5
22. ed on antibiotic resistance according to the combination of dense positive and clear no growth cell microcultures from each colony Inoculate 10 20 pl cell culture into 10 ml LB media with corresponding antibiotics Incubate in a 37 C shaking incubator overnight 13 Centrifuge the overnight cell cultures at 4000g for 5 10 minutes Purify plasmid from the resulting cell pellets with common plasmid miniprep kits according to manufacturers recommendation 14 Determine the concentrations of purified plasmid solutions by using UV absorption spectroscopy e g by using a NanoDrop 1000 machine 15 Digest 0 5 1 ug of the purified plasmid solution in a 20 ul restriction digestion with appropriate endonuclease s Incubate under recommended reaction condition for approx 2 hours 16 Use 5 10 ul of the digestion for analytical agarose 0 8 1 2 gel electrophoresis Verify plasmid integrity by comparing the experimental restriction pattern to a restriction pattern predicted in silico e g by using program VectorNTI from Invitrogen or similar programs FIEXiColi System ATG biosynthetics GmbH 33 D 5 2 Protocol 5 Deconstruction of fusion vectors by Cre The following protocol will allow you to release any single educt from multifusion constructs Cre disassembly This is achieved by Cre LoxP reaction transformation and plating on agar with appropriately reduced antibiotic resistance level cf figure 14 You can then modi
23. ents e g re ligation of the donating plasmid will be selected against by virtue of the new selector Step 2 Ligation Ligation reactions are carried out in 20 ul reaction volumes Please note that the values given below or any approximations and that success will critically depend on the correct molar ratios of FIEXiColi System ATG biosynthetics GmbH 27 vector and insert Generally a 3 1 ration insert to vector is recommended but optimum ratios may have to be determined experimentally Phosphatase treated vector x ul DNA insert y ul N x DNA Ligase buffer 20 N ul T4 DNA Ligase O 5ul 150 200 c e U or 2 4 Weiss U ddH O ad 20 ul Ligation reactions are performed at 25 C for 1 2 h or at 16 C overnight Step 3 Transformation Mixtures are next transformed into competent cells following standard transformation procedures Ligation reactions are transformed into standard E coli cells for cloning such as TOP10 DH5a HB101 and after recovery are plated on agar containing the antibiotic for which the resistance has been introduced gentamycin kanamycin chloroamphenicol Step 4 Plasmid analysis Plasmids are cultured and correct clones selected based on specific restriction digestion and or DNA sequencing of the inserts FIEXiColi System ATG biosynthetics GmbH 28 D 4 Deconstruction of gene cassette assemblies One major advantage of the Toggle system design is that you can release gene cassettes and gene as
24. ertion site using unique restriction sites Open reading frames can be inserted into the Ndel Bcll sites not shown but refer to fig 4 The ToggleTec system vectors do not contain pre integrated affinity tags for purification or solubilization of the protein s of interest Typical tags would be C or N terminal oligohistidine FLAG FIEXiColi System ATG biosynthetics GmbH 12 or myc tags or novel tags such as the stimulus responsive RTX precipitation tag Shur et al 2013 with or without protease cleavage sites for tag removal These can be introduced via gene synthesis or through specifically designed PCR primers used for amplification of the genes of interest Just follow the guidelines on critical design parameters Alternatively you can order ready to use adapted fluorescence or other tags from ATG CODING SEQUENCE nt variable Figure 8 Schematic representation of a ACDC SD gene cassette plus flanking regions in the context of the TogTec vectors By design the coding sequence is bracketed by 5 Ndel and 3 Bcll sites that contain the start ATG and stop codon TGA respectively Both restriction sites are eliminated from the coding sequence CDS and any relevant flanking sequences via computational optimization Likewise the constructionally relevant restriction sites X R1 R2 R3 R4 and Y are computationally eliminated from a gene as well although some of them are optional as they serve to swap genetic elements
25. es with a built in option for easy disassembly and substitution of sequence bits and parts This is called ACDC SC assembly cloning disassembly cloning substitution cloning design FIEXiColi System ATG biosynthetics GmbH Figure 4 Schematic representation of a gene cassette plus flanking regions to illustrate the ACDC SD principle as applied to E coli By design the coding sequence is bracketed by 5 Ndel and 3 Bcll sites that contain the start ATG and stop codon TGA respectively Both restriction sites are eliminated from the coding sequence CDS and any relevant flanking sequences via computational optimization Likewise the constructionally relevant restriction sites X R1 R2 R3 R4 R5 and Y are computationally eliminated from a gene as well although some of them are optional as they serve to swap genetic elements promoters terminators leaders etc X and Y in particular are vector specific restriction sites that demarcate the constructive borders of the A box e g Ascl and MauBl or their corresponding core sequence BssHII X and Y in figure 4 can theoretically be designed freely but the outer restriction site pair of the A box AbsI Xhol SgrDI Sall is suitable for performing the higher order concatenation of multi gene cassettes see fig 5 i Figure 5 Close up of the A box showing the outer Absl SgrDI and inner Ascl MauBl restriction site pair for toggle cloning CCIICGAGG The core sequences
26. fy and diversify the regenerated educts e g via PCR Then the altered modified construct can be reintroduced into multigene constructs via Cre LoxP reaction C3 1 4 clones LB Gentamycin Media control O LB Chloramphenicol O LB Kanamycin O LB Spectinomycin Positional marker blue dye Gm Cm Kn Sp O l ye Empty well 4 clones Figure 20 96 well analysis of Cre de assembly Reagents Cre recombinase and 10x Buffer E coli competent cells pir strains pir strains can be used only when partially deconstructed Acceptor Donor fusions are desired Antibiotics 1 Incubate approx 1 ug multifusion plasmid with 2 ul 10x Cre buffer 1 2 ul Cre recombinase add ddH O to adjust the total reaction volume to 20 ul 2 Incubate this Cre deconstruction reaction mixture at 30 C for 1 to 4 hour s 3 Optional load 2 5 ul of the reaction on an analytical agarose gel for examination Heat inactivation at 70 C for 10 minutes before gel loading is strongly recommended FIEXiColi System ATG biosynthetics GmbH 34 4 For chemical transformation mix 10 15ul De Cre reaction with 200 ul chemically competent cells Incubate the mixture on ice for 15 30 minutes Then perform heat shock at 42 C for 45 60 s Up to 20 ul De Cre reaction 0 1 volumes of the chemical competent cell suspension can be directly transformed into 200 ul chemically competent cells For electrotransformation up to 2 ul De Cre reaction can
27. h 150 ul antibiotic containing LB medium following Illustration 7 It is recommended to add coloured dye positional marker in the wells indicated A typical arrangement of the solutions which is used for parallel selections of multifusion plasmids is shown in Illustration 10 The concept behind the 96 well plate experiment is that every cell suspension from single colonies needs to be challenged by all four single antibiotics for unambiguous interpretation 11 Add 1 ul aliquots of pre incubated cell culture Step 9 to the corresponding wells Then incubate the inoculated 96 well microtiter plate in a 37 C shaking incubator overnight at 180 200 rpm Recommended use parafilm to wrap the plate to avoid drying out The remainder of the pre incubated cell cultures can be kept at 4 C for further inoculations if necessary FIEXiColi System ATG biosynthetics GmbH 32 oa A AE 4 clones 4 clones O LB Gentamycin O LB Chloramphenicol gt O LB Kanamycin Gm Cm Kn Sp O LB Spectinomycin Z B Positional marker red dye y 4 clones O Empty well Gm Kn Figure 19 96 well analysis of Cre assembly Inidividual clones from plates are transferred to wells with LB antibiotic and subjected to single antibiotic selection Clones should grow in each well for which the appropriate antibiotic resistance is hosted but fail to grow in other wells 12 Select transformants containing desired multifusion plasmids bas
28. he necessary adaptations to and the relevant biocomputational calculations for the Toggle design of your gene cassettes Please note that Bcll can only be cleaved if the plasmid DNA is amplified in dam negative strains of E coli In some configurations adequate transformed strains are included FIEXiColi System ATG biosynthetics GmbH 17 3 1 2 Inserts with Other Unique Restriction Sites If you wish to use your own set of restriction sites the Toggle design supports this as well but mandates an intermediate step for retrofitting the design For a detailed protocol please inquire at order atg biosynthetics de Alternatively you may try ligation independent procedures to introduce non standard restriction sites FIEXiColi System ATG biosynthetics GmbH 18 C 3 2 Multi gene construction via Toggle multiplication To toggle in a second gene cassette the receiving vector which can be an acceptor or donor is restricted with MauBl and AsiSl to release the selector Step 1 In parallel the donating vector is restricted with Ascl AsiSI Step 2 to release the A box S box combo which contains the second gene expression cassette The fragments can be mixed and appropriate ligation events selected by virtue of the newly introduced selector Step 3 and 4 The need for fragment purification can be obviated by dephosphorylating and additionally cleaving the donating vector with ApaLl which will destroy the ori GG CGCGCC
29. here A F Desired Acceptor Donor combinations are transformed into E coli subsequently selected by antibiotic challenge and verified by restriction PCR or DNA sequencing Correct constructs can then be amplified and isolated to transform suitable E coli expression strains LoxP sites in the acceptor donor fusions have been omitted for reasons of clarity FIEXiColi System ATG biosynthetics GmbH 15 C 3 Generating multi gene expression cassettes To create your expression constructs see illustration 3 introduce your gene or genes of interest carrying any additional modifications such as purification or reporter tags using your method of choice conventional restriction ligation cloning LIC or SLIC into any of the acceptor or donor vectors You can then create acceptor donor fusions with the help of Cre recombinase Note that you need at least one acceptor vector if you wish to amplify the multi gene constructs in standard laboratory strains Select your multi vector multi gene fusions by subjecting transformed bacteria to multiple antibiotic selection on agar and or multi well plates You will then have to extract the plasmid construct from your host strains since the expression strain will most likely be different see chapter C 5 flexeColli SAONE 2 Acceptor gt 4 Donor RAS gene s gt P TOGGLE amp gene s of interest Vectors RL P Vectors of interest Cre LoxP Recombination select E coli Expre
30. hough required or desired restriction sites can be introduced on demand Acceptors come with a gentamycin resistance marker DS Insertion Site Insertion Site T7 T7 Terminator Terminator Acc A1 5 GmR Acc A1 6 GmR 2572 bp 2623 bp a b Figure 6 Circle map representation of flexiColi acceptor vectors a TogColi A1 5 2572 bp b TogColi A1 6 2623 bp Both vectors carry a modified ColE1 origin of replication for maintenance of a high plasmid copy number Acceptor vectors host T7 TogColi A1 5 and lac TogColi A1 6 promoters T7 terminators loxP sites red circle and a gentamycin resistance marker blue Genes of interest are cloned into the unique restriction sites Ndel Bcll in the A box The multiplication module for Toggle assembly is defined by AbsI Ascl and MauBl SgrDl respectively see fig 5 Genes or gene cassettes can also be recombined via Cre Lox recombination making use of the LoxP sites hosted on the vectors Open reading frames can be inserted into the CDS insertion site framed by Ndel and Bcll see fig 4 FIEXiColi System ATG biosynthetics GmbH 11 The Donor vectors TogColiA1 5KnR GmR and TogColiA1 6 KnR GmR are similar to the acceptor vectors in their over all design but provide different selectors The multiple cloning site is bracketed by a multiplication element again Absl SgrDl or Ascl MauBl as desired to enable concatenation of inserts between the different donor vectors Vectors also contai
31. iguration from which all other designs are derived RO Figure 2 Schematic representation of the fundamental pBoX lay out R1 Toggle vectors consist of four defined constructional elements boxes application A Box selector S Box as well as target T Box and maintenance M Box functions All Boxes are separated by unique pBOX1 0 restriction sites RSs A boxes usually contain a pair of inner and outer RSs RO and RO R1 and R1 Genes and genes cassettes are cloned into the A Box which may also host other functions standard oris for plasmid propagation in E coli can be inserted exchanged in the maintenance box whereas the T box will carry oris for specific applications or target organisms the S box hosts the selector functions most commonly antibiotic resistance markers The TogTEC systems are characterized by a unique constallation of restriction sites RSs that demarcate the four Boxes figure 3 Figure 3 Implementation of pBoX in TogTEC vectors All TogTec vectors share a common lay out where the A Box is bracketed by Absl and Ascl at the 5 end and MauBI SgrDI at the 3 end The S Box is demarcated by SgrDI and AsiSI while the T Box and M Box are defined by AsiSl Pacl and Pacl Absl respectively Vector The functional role of the individual Boxes is identical to pBoX Along with this slender make up the vectors are designed to allow highly flexible generation of multigene assembli
32. ing to the combination of dense growth and clear no growth cell cultures from each colony Inoculate 10 20 ul from the cell cultures into 10 ml LB media with corresponding antibiotic s Incubate in a 37 C shaking incubator overnight The next day centrifuge the overnight cell cultures at 4000g for 5 10 minutes Purify plasmid from cell pellets with common plasmid miniprep kits according to manufacturers protocols Determine the concentrations of purified plasmid solutions by using UV absorption spectroscopy e g NanoDrop 1000 Digest 0 5 1 ug of the purified plasmid solution in a 20 ul restriction digestion with 5 10 units endonuclease Incubate under recommended reaction condition for approx 2 hours Use 5 10 ul of the digestion for analytical agarose gel 0 8 1 2 electrophoresis Verify plasmid integrity by comparing the de facto restriction pattern to the in silico predicted restriction pattern e g by using VectorNTI Invitrogen or any other similar program Optional Occasionally a deconstruction reaction is not complete but yields partially deconstructed fusions which still retain entities to be eliminated In this case we recommend to pick these partially deconstructed fusions containing and perform a second round of Cre deconstruction reaction repeat steps 1 8 by using this construct as starting material FIEXiColi System ATG biosynthetics GmbH 36 D 6 Expression of multi gene constructs This will not be c
33. ment Figure 1 Applications of E coli multi protein expression systems Some of these are closely intertwined e g pathway design metabolic engineering and strain development Escherichia coli is your first choice for expressing proteins and protein complexes that do not require extensive complex posttranslational modifications E coli has proved its worth time and again as the go to organism for initial investigations While E coli has some disadvantages such as the limits in protein sizes one can express the problem of protein being sequestered into inclusion bodies or the inablity to handle complex glysosylation there are many advantages that make it the favorite workhorse for innumerable expression projects These include the wide range of gene engineering tools that allow its genetic modification the ease of culturing Escherichia coli various induction protocols rapid growth etc FIEXiColi System ATG biosynthetics GmbH C A Novel Approach to Multigene Applications C 1 The Toggle Concept a Primer TogTec Vectors represent a novel stringent vector design that aims at clearly trimmed vector backbones that still provide all required functionality This can be achieved by stripping all elements to the bare minimum necessary for proper function Toggle systems consists of four boxes that are reserved for and harbor different modular functions as exemplified by the pBoX vector in figure 2 It represents the archetypical conf
34. n a LoxP imcomplete inverted repeat to create acceptor donor or donor donor fusions The vectors are equipped with a kanamycin resistance marker but can be outfitted with any other resistance by exchanging the S box Donor vectors are traditionally defined by a conditional R6Ky origin of replication which makes its propagation dependent on the expression of the pir gene in the prokaryotic host such as the pirLC and pirHC cells contained in the kit Yet TogTec vectors can be turned from acceptors with a constitutive standard bacterial origin of replication ori into donors with a conditional ori via a conversion protocol page 24 LoxP T7Promoter LoxP 7 promoter CDS Insertion Site CDS Insertion Site T7 Terminator T7 Terminator fLEXColi fLEXColi Don A1 5 ciR Don A1 5 KnR 2462 bp 2584 bp LoxP lac Promoter LoxP jac Promoter CDS Insertion Site CDS Insertion Site T7 Terminator T7 Terminator flLEX Coli flLEX Coli Don A1 6 CIR Don A1 6 KnR 2513 bp crap C d Figure 7 Circle map representation of flexiColi donor vectors a c TogColi A1 5 CmR 2462 bp KnR 2584 bp b d TogColi A1 6 CmR 2513 bp KnR 2635 bp Circle maps show promoters T7 lac terminators T7 multiplication module the incomplete inverted repeat for cre lox site specific recombination LoxP orange circle and resistance markers chloroamphenicol and kanamycin neomycin respectively Genes of interest are cloned into the multiplication module CDS ins
35. ocol 5 Deconstruction of fusion vectors by Cre D 6 Expression of multi gene constructs E Appendix E 1 Preparing chemically competent cells 10 15 16 16 17 18 20 22 23 23 23 24 24 24 25 28 28 30 30 33 36 37 37 FIEXiColi System ATG biosynthetics GmbH E 2 Preparing bacterial stocks from agar stabs E 3 flEXiColi vectors maps sequences restriction E 3 1 Acceptor vectors E 3 1 1 TogColiAccA1 5 amp A1 6 2572 bp 2623 bp E 3 2 1 TogColiDonA1 5 2774 bp KnR 2619 bp CmR E 3 2 2 TogColiDonA1 6 2864 bp KnR 2693 bp CmR E 3 3 Analytical restriction digest patterns E 3 4 Sequencing primers F References NOTES G Purchaser Notification FIEXiColi System 38 39 40 40 41 42 43 43 45 46 47 ATG biosynthetics GmbH A ALEXcColi kit contents e Plasmid acceptor transfer vectors TogColiA1 5GmR TogColiA1 6GmR approx 3 4 ug DNA per vial in buffer solution keep at 4 C for short term storage and in a freezer at 20 C or lower for medium and long term storage take care to avoid repeated freeze thaw cycles e g by aliquotting DNA prior to freezing e Plasmid donor vectors TogColiA1 5KnR TogColiA1 5CmR TogColiA1 6KnR amp TogColiA1 6CmR approx 3 4 ug DNA per vial in buffer solution keep at 4 C for short term storage and in a freezer at 20 C or lower for medium and long term storage take care to avoid repeated freeze thaw cycles e g by aliquotting DNA
36. ombineering The resulting multigene fusions can be transformed into specific expression host strains This protocol can be used to integrate multigene cassettes with coding sequences for multiprotein complex subunits into flexiColi but also to integrate specific enzymes kinases acetylases etc for modifying the proteins under investigation 4 Individual gene cassettes used in the Toggle system can in fact be re cycled and re used to build other multi gene constructs This will facilitate building multigene expression constructs with permutated composition or shuffling the order of expression cassettes to probe any positional effects FIEXiColi System ATG biosynthetics GmbH 14 TTec Acceptor TTec Donor AAA AA TlecAcc ABC TlecDon DEF Cre Lox site specific recombination in vitro Electroporation heat shock transformation Acceptor strain Expression strain Induction and Protein Expression Purification Figure 9 Schematic overview of the workflow of the flexiColi system Genes of interest are assembled into multigene expression cassettes using either gene synthesis the Toggle multiplication module present on the donor and acceptor vectors or any of several other methods the ToggleTec vectors support Acceptor donor multigene fusions can also be generated by Cre LoxP recombination In this case the multigene fusions contain one Acceptor and one to several Donor vectors each with one or several genes of interest
37. only have a limited shelf life 3 12 months depending on storage conditions Permanent cultures in glycerol or DMSO on the other side should yield viable colonies even after 1 year and beyond if properly stored To generate your bacterial stock for long term storage streak bacteria from the agar stab onto an appropriate selective plate refer to the vector maps for acceptor and donor vectors or plates without antibiotics pirHC and pirLC strains we recommend to test these strains against a panel of antibiotics to be on the safe side no growth of colonies should be observed under conditions of antibiotic selection Incubate the plates over night at 37 C and then proceed to prepare stocks from individual colonies for long term storage according to your protocol of choice glycerol DMSO etc as decribed for example in Inoue et al 1990 Molecular Cloning Sambrook and Russell 2000 Current Protocols in Molecular Biology Ausubel et al 1994 etc FIEXiColi System ATG biosynthetics GmbH 39 E 3 flEXiColi vectors maps sequences restriction Note All acceptor and donor vector sequences can be provided in electronic format as files compatible with the ApE A plasmid Editor currently July 2013 v2 0 46 sequence analysis and presentation software These sequences contain all relevant information such as unique restriction sites oris resistance markers etc that is also shown in the circle maps Request your set of vector
38. overed in the manual as we assume that experimenters have a working knowledge of suitable strains and growth induction conditions The Current Protocols in Molecular Biology Ausubel et al and the article by Tolia and Joshua Tor 2006 should be excellent starting points and references for protein expression in bacterial systems Specific strains and applications are presented and discussed among others in Valdez Cruz et al 2010 Lobstein et al 2012 FIEXiColi System ATG biosynthetics GmbH 37 E Appendix E 1 Preparing chemically competent cells Aside from the protocol provided below there are numerous protocols for preparing competent cells for heat shock transformation e g the classical CaCl method the TSS protocol originally by Chung et al 1989 PNAS USA Vol 86 pp 2172 2175 etc You will find various protocols online e g http francois schweisguth free fr protocols TSS competent cell preparation pdf http www personal psu edu dsg11 labmanual DNA manipulations Comp bact by TSS htm http openwetware org wiki Preparing chemically competent cells or in Sambrook and Russell Preparing competent E coli TOP10 cells with the CaCl method 1 Grow E coli strain by streaking on LB agar plates incubate o n at 37 C 2 Growa few colonies in 20ml LB at 37 Co n to OD 600 1 8 3 Culture 13ml into 100ml freshly prepared LB to OD 600 0 25 4 Incubate at 37 Con a shaker 180 rpm for 1 hou
39. prior to freezing e E coli strains as agar stabs opt in component a transformed with acceptor and donor vectors 6 vials For streaking bacteria on plates as a starting point for plasmid preparation b optional in some configurations pirHC pirLC cells For propagation and amplification of donor vectors donor multi gene expression constructs or donor donor fusions Keep agar stabs at 4 C or at RT do not freeze We recommend to immediately prepare stocks from streaked bacterial colonies see p 38 E coli strains expressing the pir gene for propagation of donor vectors any other strain with pir background can be used as well LC low copy number propagation HC high copy number propagation of plasmids with R6Ky origin FIEXiColi System ATG biosynthetics GmbH Reagents to be supplied by user see also section D Protocols e Restriction endonucleases and other DNA modifying enzymes e TZ or any other suitable DNA ligase e Crerecombinase e Standard E coli strains for cloning such as TOP10 DH5a HB101 etc e Standard laboratory buffers solutions media and equipment for bacterial cell culture transformation etc e Commercially available transfection reagents e g FUGENE Roche jetPEI Polyplus transfection GeneJuice etc alternatively equipment for electroporation e Antibiotics chemicals e g IPTG X Gal etc e pir cells depending on the kit configuration FIEXiColi System ATG biosyn
40. promoters terminators leaders etc X and Y in particular are vector specific restriction sites that demarcate the constructive borders of the A box e g Ascl and MauBl or their corresponding core sequence BssHII This E coli expression system offers a few re designs and improvements that will help in fully exploiting the potential of this heterologous protein production system 1 New transfer vectors TogColiA1 5 TogColiA1 6 TogColiA1 5 TogColiA1 6 see figures 6 and 7 that adhere to the uniform ToggleTec vector design Gene concatenation multiplication is achieved via directional cloning into unique restriction sites of the multiplication module These vectors greatly facilitate modular combination of heterologous genes in their respective gene expression cassettes FIEXiColi System ATG biosynthetics GmbH 13 with a minimum requirement for unique restriction sites Strong bacterial promoters currently T7 and lac very late prmoters can be exchanged for other promoter sequences constiutive and inducible if desired Likewise terminator sequences currently T7 can be substituted as required as schematically shown in fig 4 2 Leaders for T7 and lac promoters possess a uniform length This obviates the inconsistencies produced by multiple cloning sites that add a variable number of nucleotides to the leader promoter 3 Established protocol for rapid generation of multigene expression constructs via Cre LoxP rec
41. r OD600 0 5 0 6 depending on the strain 5 Distribute the culture into 50ml centrifuge tubes and centrifuge at 8000 rpm 4 C for 5 min or alternatively in 50 ml Falcon tubes spin at 4000rpm for 6 min 6 Discard the supernatant completely then add 10ml of ice cold 0 1M CaCl keep the cells on ice and resuspend gently 7 Incubate the solution on ice for 15 min then centrifuge at 8000 rpm at 4 C for 5 min or alternatively in 50 ml Falcon tubes spin at 4000rpm for 6 min 8 Remove the supernatant completely and let the pellet air dry 9 Add 2 8ml ice cold 0 1M CaCl and mix gently Keep on ice till use OR 10 Add 1 2 ml of sterile ice cold 80 glycerol to a tube with 2 8ml cells each and mix gently transfer 100 ul aliquots into sterile 1 5 ml Eppendorf microcentrifuge tubes use gloves and Bunsen burner flame keep the Eppendorfs in ice until snap freezing the bacterial cells in liquid N store at 80 C Alternatively transfer 100 ul aliquots into sterile 1 5 ml Eppendorfs add 32 ul of 80 glycerol freeze in dry ice EtOH bath or liquid N and store at 80 C FIEXiColi System ATG biosynthetics GmbH 38 E 2 Preparing bacterial stocks from agar stabs We recommend preparing your personal bacterial stock from the agar stabs you received in the kit or transforming your laboratory strain of choice with the vectors please note that for the donor vectors this needs to be a pir strain This is advisable since agar stabs
42. ral different single educts partially deconstructed multifusion plasmids can be processed and selected in parallel on one 96 well microtiter plate 9 For 96 well analysis inoculate four colonies each from agar plates containing a defined set of antibiotics into approx 500 ul LB medium without antibiotics Incubate the cell cultures in a 37 C shaking incubator for 1 2 hours 10 While incubating the colonies fill a 96 well microtiter plate with 150 ul antibiotic containing LB medium or dye positional marker in the corresponding wells Refer to figures 10 and 11 for the arrangement of the solutions in the wells which are used for parallel selection of single educts or partially deconstructed multifusion plasmids The concept is that every cell suspension from a single colony needs to be challenged by all four antibiotics separately for unambiguous interpretation FIEXiColi System ATG biosynthetics GmbH 11 12 13 14 15 16 17 35 Add 1 ul aliquots from the pre incubated cell cultures Step 9 into the corresponding wells Incubate the 96 well microtiter plate in a 37 C shaking incubator overnight at 180 200 rpm Recommended use parafilm to wrap the plate to prevent desiccation The remainder of the pre incubated cell cultures can be kept at 4 C in a refrigerator for further inoculations if necessary Select transformants containing desired single educts or partially deconstructed multifusion plasmids accord
43. rnative protocol established in your lab Elution of the extracted DNA in the minimal volume defined by the manufacturer is recommended The vector backbone should be treated with DNA phosphatase alkaline calf intestinal shrimp alkaline antarctic according to the suppliers recommendations Dephosphorylation is performed to minimize vector re annealing and to increase integration of the insert Vector backbone DNA in ddH20 buffer x ul N x phosphatase buffer 25 N ul DNA phosphatase 1ul ca 10U Alternatively you can omit the agarose gel electrophoresis and purification step by simply precipitating the DNA from both restriction digestions and then dephosphorylating the vector backbone receiving plasmid In this case antarctic phosphatase may be your best choice as it is reliably inactivated at 65 C after 5 minutes Residual phosphatase activity is believed to interfere with the ligation reaction As an additional measure you can digest the donating plasmid with ApaLl which will destroy the ColEl ori in the residual plasmid backbone If the backbone should reassemble during cloning chances are good it will be as a conditional R6Ky ori which is selected against in standard subcloning strains of E coli You can then simply mix appropriate ratios of DNA from both reactions and perform the ligation reaction The cloning efficiency will likely be reduced but this approach should generate a sufficient number of clones as any incorrect ligation ev
44. rom NEB or self made Standard E coli competent cells pir strain Antibiotics 96well microtiter plates if required 12 well tissue culture plates or petri dishes w agar antibiotics LB medium 1 Fora 20ul Cre reaction mix 1 2 ug of each educt in approximately equal amounts Add ddH O to adjust the total volume to 16 17 ul then add 2 ul 10x Cre buffer and 1 2ul Cre recombinase 1 2 U 2 Incubate Cre reaction at 37 C or 30 C for 1 hour 3 Optional load 2 5 ul of Cre reaction on an analytical agarose gel for examination Heat inactivation at 70 C for 10 minutes before gel loading is strongly recommended 4 For chemical transformation mix 10 15ul Cre reaction with 200 ul chemically competent cells Incubate the mixture on ice for 15 30 minutes Then perform heat shock at 42 C for 45 60 s Up to 20 ul Cre reaction 0 1 volumes of the chemically competent cell suspension can be directly transformed into 200 ul chemically competent cells For electrotransformation up to 2 ul Cre reaction can be directly mixed with 100 ul electrocompetent cells and transformed by using an electroporator e g BIORAD E coli Pulser at 1 8 2 0 kV Larger volumes of Cre reaction must be desalted by ethanol precipitation or via PCR purification columns before electrotransformation The desalted Cre reaction mix should not exceed 0 1 volumes of the electrocompetent cell suspension The cell DNA mixture can be immediately used for ele
45. semblies for use in other multi gene cloning projects by simple restriction digestions As the restriction sites are compatible with the entire design you can use them to build a different design D 4 1 Protocol 3 Disassembling multi gene construct to regenerate individual expression boxes Expression cassettes hosted in A boxes can be regenerated by 1 digesting the multi gene construct with Ascl SgrDlI Xhol Sall complete A box including LoxP site fig 17A or Ascl MauBl multigene cassette including promoter gene of interest and terminators fig 17B to release the entire assembly The overhangs created by either pair of restriction enzymes are identical i e apolar see figure 17 Restricting with Ascl SgrDl on the other hand will create incompatible overhangs that confer polarity onto the fragment fig 17C CC TCGAGG CG CGCGCG Ascl CG CGCGCG SgrDI CG TCGACG SgrDI CG TCGACG Figure 17 Releasing individual genes or gene assemblies from TogTec vectors General lay out of TogColi donor vectors A serves for clearer illustration as they lack the Tn7L and Tn7R sequences Note that you have an outer pari of restriction sites Absl SgrDl and an inner pair Ascl MauBl which are compatible in terms of yielding identical overhangs Restriction digest with B Absl MauBDl C Ascl MauBl D Absl SgrDI and E Ascl SgrDI yield the results described in the main text While Absl SgrDl and Ascl MauBl generate apolar overhangs Absl
46. ss proteins protein complexes Figure 10 Schematic representation of process for generating multi gene expression constructs If for example your requirements for an antibiotic resistance marker change you can transfer entire expression cassettes including promoters and terminators within acceptors and donors and between acceptors and donors by using the Absl SgrDI Xhol Salll or Ascl MauBl BssHII module see fig 17 For expression in E coli the ACEXColi multi gene expression vector fusions with appropriate promoters or terminators are transformed into the appropriate expression host of choice The basic version T7 and lac promoter elements should support expression in most currently available expression strains If particular expression strains already contain helper plasmids with DNA encoding chaperones lysozyme or other factors of interest the design of the multi gene fusion should ideally be such that the AEMAColi vector containing the resistance marker that is also present on the helper FIEXiColi System ATG biosynthetics GmbH 16 plasmid is not included in multi gene vector construction to avoid issues with plasmid incompatibility although this is probably not essential Alternatively the issue can be resolved by creating new versions of the AEXColi vectors containing resistance markers that circumvent the conflict This can be easily performed by PCR amplifying the vectors minus the resistance marker
47. t is not limited to 1 use of the product or its components in manufacturing 2 inclusion of the product as part of another product 3 use of the product or its components to provide a service information or data 4 use of the product or its components for ex vivo or in vivo therapeutic diagnostic prophylactic or other unauthorized commercial purposes 5 use in foods drugs devices or cosmetics of any kind or for consumption by or use in connection with or administration or application to humans or animals 6 resale of the product or its components whether or not such product or its components are resold for use in research or 7 any use other than research use e g any formalized in house screening programs that are not for ealuation purposes For profit Industry Customers As detailed in a separate agreement ATG will grant to the Licensee a nonexclusive limited term usually one year license for evaluation research and development purposes only within its research facilities to make and use but not to sell the Materials or the Licensed Products and products and processes encompassed within the scope of a claim in the Licensed Patent Rights The Licensee agrees that any commercial or industrial use or sale of any such products or processes including other than for evaluation purposes shall be made only pursuant to the terms of a commercialization license to be negotiated in good faith by the parties For products that
48. thetics GmbH B Escherichia coli a premier laboratory workhorse B 1 Introduction Protein complexes are the heart and soul of many crucial cellular processes Robinson et al 2007 Charbomnier et al 2008 Some researchers go as far as describing the cell as a collection of protein machines Albert 1998 which in essence are multi protein complexes Whether you think of replication transcription Van Hijum et al 2009 translation Estrozi et al 2011 DNA repair the processing import trafficking as well as export of proteins or other biomolecules or the maintenance of the structural stability and integrity of any cell multi subunit protein assemblies play an important role in all these biological phenomena In addition other processes e g entry of viruses into human cells also critically hinge on the co operation of proteins or protein complexes Bhattacharya 2009 Various prokaryotic microorganisms E coli being the prototypical workhorse are harnessed to express heterologous proteins and protein complexes but also to cost efficiently produce known or novel compounds by means of metabolic engineering Chamler and Koffas 2008 Chou 2007 Lee et al 2008 Scientists wishing to study these processes in functional and structural detail often require significant amounts of the protein complexes under investigation While obtaining bulk protein usually is not a problem for protein complexes that are abundant in
49. ts with 5 Ndel and 3 Bcll restriciton sites or b amplified from existing sources with PCR primers hosting a Ndel for the 5 terminus of the forward primer and a Bcll for the 3 terminus of the reverse primer restriction site while making sure that the ORF does not contain any of the restriction sites listed on page 13 Ligation reactions for TogColi acceptor vectors are transformed into standard E coli cells for cloning such as TOP10 DH5a HB101 and plated on agar containing gentamycin 7 ug ml Ligation reactions for TogColi donor vectors are transformed into a standard E coli cells or b into those expressing the pir gene if the conversion of the ori has been carried out The strain may be pirHC and pirLC from this kit in this case you will need to make the cells electro or chemically competent first or any other commercially available strains e g BW23473 BW23474 Next plate putative transformants on agar containing chloramphenicol 25 ug ml or kanamycin 50 ug ml Correct clones are selected based on specific restriction digestion patterns and DNA sequencing of the inserts FIEXiColi System ATG biosynthetics GmbH 24 D 2 Conversion of donors into conditional donors Donor vectors TogColiA1 5 and TogColiA1 6 host split origins of replication that in their default state are ColE1 derived oris If desired and required they can be converted into conditional pir dependent oris by following this conversion proto
50. ue H Nojima H and Okayama H 1990 High efficiency transformation of Escherichia coli with plasmids Gene 96 23 28 Lee SK Chou H Ham TS Lee TS and Keasling JD 2008 Metabolic engineering of microorganisms for biofuels production from bugs to synthetic biology to fuels Current Opinion in Biotechnology 19 556 563 Li M and Elledge SJ 2007 Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC Nature Methods 4 251 256 Lobstein J Emrich CA Jeans C Faulkner M Riggs P and Merkman M 2012 SHuffle a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm Microbial Cell Factories 11 56 Robinson CV Sali A and Baumeister W 2007 The molecular sociology of the cell Nature 450 973 982 Sambrook J and Russell DW 2000 Molecular Cloning A Laboratory Manual 3rd edition Cold Spring Harbor Laboratory Press Cold Spring Harbor available online at http cshprotocols cshlp org Tan S Kern RC and Selleck W 2005 The pST44 polycistronic expression system for producing protein complexes in Escherichia coli Protein Expression and Purification 40 385 395 Tolia NH and Joshua Tor L 2006 Strategies for protein coexpression in Escherichia coli Nature Methods 3 55 64 Valdez Cruz NA Caspeta L P rez NO Ramirez OT Trujillo Roldan MA 2010 Production of recombinant proteins in E coli by the heat inducible expression system based
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