Direct cloning-proficient E. coli Strain GB05-dir
Contents
1. Cat Na KOOS CT III THE RECOMBINEERING COMPANY ENE BRIDBGES LLS Direct cloning proficient E coli Strain GBO5 dir pa os vv a Fo r Red ET Recombination m m nn o _ o ee A O ie _ NN LLL LLL a dr _ ee A nn _ Y as A ee ee EE ee KIKO ee n M A A nn mm e pana 0 a ijj 12 Version 1 1 May 2014 CONTENTS 1 Direct cloning proficient E coli Strain GBO5 dir 2 n22aannn000nonnnnnnnnnnnnnnonnnnnnnnnnnnnn 2 EXperimental OUTLET zen EEE een 3 How Red ET Recombination W rkS un na nnmnnn nnmnnn 4 TESCHNIEAL o AAPP sen reece tends nennen genen Era ee EA 4 1 Generation of a vector backbone flanked by homology arms uus22222000nenne nennen 4 2 Assembly of a linear DNA insert and a linear plasmid backbone s 2 220022220 no 4 3 Verification of successfully modified plasmid by restriction analysis 222220s2222200 4 4 Maps and SEOLENEES nn ee 5 TroubleshoOl O ces secede aes sauce E E A 6 FICTEIENCES and Patent 10 6 1 ROTOS nn a ne ee 11 6 2 FINS tad ee ren en obres 11 7 Purchaser Notification Warranty cu nein na nenn nn een 11 8 O2. 12 Gene Bridges Direct cloning proficient E coli Strain GBO5 dir Version 1 1 May 2014 8 Other products available from Gene Bridges General information e Kits are available for non commercial research organizations Commercial companies or for profit organizations require a sub license to use Red ET Recombination The complete product list as well as all information about how to order kits in your country is given on our website www genebridges com Additional Kits e K001 Quick and Easy BAC Modification Kit e K002 Counter Selection BAC Modification Kit e K003 BAC Subcloning Kit e K004 Quick and Easy Conditional Knockout Kit FRT FLPe e K005 Quick and Easy Conditional Knockout Kit loxP Cre e K006 Quick and Easy E coli Gene Deletion Kit e K009 Recombineering proficient E coli strain GBO8 red Additional functional cassettes e A001 Pro and Eukaryotic Neomycin Selection Cassette PGK gb2 neo e A002 FRI flanked Pro and Eukaryotic Neomycin Selection Cassette FRT PGK gb2 neo FRT e A003 loxP flanked Pro and Eukaryotic Neomycin Selection Cassette loxP PGK gb2 neo loxP e A004 FRI flanked Pro and Eukaryotic Neomycin Selection Cassette plus loxP site FRT PGK gb2 neo FRT loxP e A005 FRI flanked Pro and Eukaryotic Neomycin Selection Cassette plus loxP site 2 version loxP FRT PGK gb2 neo FRT e A006 FRT flanked Chloramphenicol Selection Cassette FRT cm FRT e A007 loxP flanked Chloramp
3. e Thirty cycles 1 95 1 57 62 C 2 5 72 C 6 Gene Bridges Direct cloning proficient E coli Strain GBO5 dir Version 1 1 May 2014 4 2 Assembly of a linear DNA insert and a linear plasmid backbone In the next step prepare electro competent cells from strain GBO5 dir shortly after inducing the expression of the recombination proteins In advance prepare the linear DNA fragment and the plasmid backbone with homology arms Use tube 2 control plasmid backbone and tube 3 control PCR product to perform a control experiment in parallel 1 Inoculate 1 0 ml LB medium without addition of antibiotics with GBO5 dir and incubate the culture over night at 37 C Before starting the next day e Chill ddH20 or 10 glycerol on ice for at least 2 h e Chill electroporation cuvettes 1 mm gap e Cool benchtop centrifuge to 2 C Set up 4 lid punctured microfuge tubes 2 for your own experiment and 2 for control experiment containing 1 4 ml each of fresh LB medium and inoculate two of them with 30 ul fresh overnight culture for your experiment the other two with 30 ul of the overnight culture from the control experiment Incubate the tubes at 37 C for 1 5 h shaking at 1100 rpm until OD600 0 3 Add 50 ul 10 L arabinose to one of the tubes for your own experiment and to one of the control tubes giving a final concentration of 0 3 0 4 This will induce the expression of the Red ET Recombination proteins Do not use D
4. arabinose Leave the other tubes without induction as negative controls Incubate all at 37 C shaking for 35 minutes Remark Prepare a 10 L arabinose Sigma A 3256 stock solution in ddH20 and use it fresh or frozen in small aliquots at 20 C Frozen aliquots should not undergo more than three freeze thaw cycles Prepare the cells for electroporation Centrifuge for 30 sec at 11 000 rpm ina cooled microfuge benchtop centrifuge at 2 C Discard the supernatant by quickly tipping it out twice and place the pellet on ice Resuspend the pellet with 1 ml chilled ddH2O or 10 glycerol pipetting up and down three times to mix the suspension Repeat the centrifugation and resuspend the cells again Centrifuge and tip out the supernatant once more 20 to 30 ul will be left in the tube with the pellet Keep the tube on ice Add 100 ng of the linear fragment and 100 ng of the linear plasmid backbone to each of the two microfuge tubes induced and uninduced and pipette the mixture into the chilled electroporation cuvette In parallel pipette 1 ul from tube 2 and 3 into each of the two tubes of the control Electroporate at 1350 V 10 uF 600 Ohms This setting applies to an Eppendorf Electroporator 2510 using an electroporation cuvette with a slit of Gene Bridges Direct cloning proficient E coli Strain GBO5 dir Version 1 1 May 2014 7 1mm Other devices can be used but 1350 V and a 5 ms pulse are recommended 7 Add 1 ml LB m
5. C PUL 25 000 20 7 1 500 E 1 000 100 BOD 30 G00 60 400 10 200 20 Figure 3 Restriction analysis of 6 clones obtained from the control reaction lanes 1 6 after EcoRV digestion M Hyperladder Bioline 8 Gene Bridges Direct cloning proficient E coli Strain GBO5 dir Version 1 1 May 2014 4 4 Maps and sequences u a fj e ca ie recT gam recA Figure 4 Diagram of the arabinose inducible ETyA operon full length recE recT redy and recA inserted at the ybcC locus 5 Troubleshooting Problems with the recombination reaction can be caused by a number of different factors Please review the information below to troubleshoot your experiments We highly recommend performing a positive control experiment using the components provided in the kit For homologous recombination the two DNA molecules must share two regions of perfect sequence identity Wrong nucleotides in the homology region can completely abolish recombination Since oligonucleotides are used to add the homology regions they have to be synthesized properly and be of excellent quality Take into account that long oligonucleotides especially if they are longer than 80bp require additional purification steps such as HPLC Also note that the electronic sequences provided from databases may not be 100 correct If you are trying to target a repeated sequence in your BAC or plasmid you may experience problems because th
6. Stewart A F Zhang Y and Muyrers J P P 1999 Methods and compositions for directed cloning and subcloning using homologous recombination European Patent No 1204740 United States Patent No 6355412 e Zhang Y Fu J and Stewart A F 2011 Direct Cloning WO2011 154927 These patents and patent applications are owned by Gene Bridges or owned by the EMBL and exclusively licensed to Gene Bridges 7 Purchaser Notification Warranty The purchase of this product conveys to the purchaser the non transferable right to use this product for research purposes only The purchaser cannot sell or otherwise transfer this product or its components to a third party No rights are conveyed to the purchaser to use this product or its components for a commercial purpose Commercial purposes shall include any activity for which a party receives consideration of any kind These may include but are not limited to use of the product or its components in manufacturing to provide a service information or data use of the product for diagnostic purposes or re sale of the product or its components for any purpose commercial or otherwise Limited Warranty Gene Bridges is committed to providing customers with high quality goods and services Gene Bridges assumes no responsibility or liability for any special indirect incidental or consequential loss or damage whatsoever This warranty limits Gene Bridges GmbH s liability only to the cost of the product
7. ches of DNA shared by the two molecules that recombine Because the sequence of the homology regions can be chosen freely any position on a target molecule can be specifically altered Red ET recombination allows you to choose homology arms as short as 50 bp for homologous recombination which can easily be added to a functional cassette by long PCR primers Full length Rac prophage protein RecE and its partner RecT mediate highly efficient linear to linear homologous recombination mechanistically distinct from conventional recombineering mediated by Reda from lambda phage Fu et al 2012 E coli strain GBO5 dir harbours an arabinose inducible ETgA operon full length recE recT redy and recA at the ybcC locus The strain was successfully used to directly clone gene clusters larger than 10 kb of secondary metabolite pathways from Photorhabdus luminescens a gram negative rod shaped bacterium into expression vectors Fu et al 2012 Contents of the kit 1 GB05 dir Glycerol stock of recombineering proficient E coli strain GB05 dir 500 ul 25 glycerol 2 Linear control plasmid backbone plasmid backbone containing a ColE1 origin of replication and an ampicillin resistance marker gene 100 ng ul 10 ul 3 Linear control insert PCR product linear DNA fragment consisting of a kanamycin resistance marker gene flanked by 50 bp long homology arms for recombination into the control backbone 100 ng ul 10 ul 4 This manual with p
8. d ET Recombination works Target DNA molecules are precisely altered by homologous recombination in E coli cells which express the phage derived protein pairs either RecE RecT from the Rac prophage or Reda Red from A phage These protein pairs are functionally and operationally similar RecE and Reda are 5 3 exonucleases and RecT and Red are DNA annealing proteins A functional interaction between RecE and ReclT or between Reda and Red is also required in order to catalyse the homologous recombination reaction Recombination occurs through homology regions which are stretches of DNA shared by the two molecules that recombine Figure 2 The recombination is further assisted by A encoded Gam protein which inhibits the RecBCD exonuclease activity of E coli Double stranded break 3 5 RecE 5 3 gt or Redo exonuclease gt 3 3 OOOOO nee Single strand in binding proteins 9 3 3 Joint molecule formation DNA replication Figure 2 Mechanism of Red ET Recombination Gene Bridges Direct cloning proficient E coli Strain GB05 dir Version 1 1 May 2014 5 Double stranded break repair DSBR is initiated by the recombinase protein pairs RecE RecT or Reda RedB First RecE or Reda digests one strand of the DNA from the double stranded break leaving the other strand as a 3 ended single stranded DNA overhang Then RecT or Red binds and coats the single strand The protein nucleic ac
9. e homology region at the end of the linear fragment can target more than one site Gene Bridges Direct cloning proficient E coli Strain GBO5 dir Version 1 1 May 2014 9 Observation No colonies on your plate after Red ET Recombination If you do not obtain any colonies after recombination the following parameters should be checked 1 The linear DNA fragments PCR products o could be wrong check it by restriction digest or sequencing o could be degraded check an aliquot on an agarose gel o could have incorrect homology arms if possible verify the correct homology arms by sequencing 2 The Red ET reaction did not take place because o noor the wrong type of arabinose was used for induction please make sure you use L arabinose o in very rare cases increasing the time for recombination from 70 min incubation after electroporation to up to four hours is necessary for successful recombination 3 Problems with and after the electroporation o cells are not competent enough to take up the linear DNA Please make sure that the cells were kept on ice and that the water or 10 glycerol is sufficiently cold Linear DNA has been shown to be about 10 fold less active than DNA transformed in circular form Eppendorf Operation Manual Electroporator 2510 version 1 0 Make sure that the time constant is around 5 ms o make sure that there is no arching during the electroporation process o make sure that after electropo
10. edium without antibiotics to the cuvette Mix the cells carefully by pipetting up and down and pipette back into the microfuge tube Incubate the cultures at 37 C with shaking for about 90 min Recombination will now occur 8 Streak the cultures with a loop onto LB agar plates containing the appropriate antibiotics for the plasmid e g ampicillin 100 pg ml for the control You should obtain gt 100 colonies and the ratio of induced uninduced bacterial colonies should exceed 10 1 More than 95 of all colonies growing on the agar plates conditioned with the appropriate antibiotics will have successfully recombined copies of the plasmid Please note that although most ampicillin resistant colonies will contain the correct plasmid recombinant in rare cases it is possible that secondary recombination usually deletions between internal repeats in the plasmid can also occur To confirm the correct recombination event pick 10 20 colonies from your experiment and 2 from the control reaction isolate plasmid DNA and analyze the DNA by restriction digestion 4 3 Verification of successfully modified plasmid by restriction analysis Analyze an aliquot of your plasmid DNA by restriction digestion For the control experiment the restriction pattern after EcoRV digestion is 2641bp 1741bp Figure 3 1 N O IN al O M BAND SIZE bp ng BAND e y i E gt 2 e i 50 Ey m 000 a
11. henicol Selection Cassette loxP cm loxP e A008 FRT flanked Ampicillin Selection Cassette FRT amp FRT e A009 loxP flanked Ampicillin Selection Cassette loxP amp loxP e A010 FRI flanked Pro and Eukaryotic Hygromycin Selection Cassette FRT PGK gb2 hygro FRT Gene Bridges Direct cloning proficient E coli Strain GBO5 dir Version 1 1 May 2014 13 e A011 loxP flanked Pro and Eukaryotic Hygromycin Selection Cassette loxP PGK gb2 hygro loxP e A012 FRT flanked Pro and Eukaryotic Neomycin Selection Cassette with attached codon improved Cre iCre FRT PGK gb2 neo FRT Additional strains and plasmids A104 Enhanced FLP Expression Plasmid 707 FLPe with tetracycline tet resistance marker for use in E coli only A105 Enhanced FLP Expression Plasmid 708 FLPe with chloramphenicol cm resistance marker for use in E coli only A106 Enhanced FLP Expression Plasmid 709 FLPe with ampicillin ap resistance marker for use in E coli only A107 Enhanced FLP Expression Plasmid 710 FLPe with kanamycin km resistance marker for use in E coli only A112 Cre Expression Plasmid 705 Cre cm resistance marker A113 Cre Expression Plasmid 706 Cre tet resistance marker A201 Enhanced Eukaryotic FLP Expression Plasmid PCAGGS FLPe 9 DNA Engineering Services Available from Gene Bridges Instead of performing your own DNA manipulations let the Gene Bridges DNA Engineering Service Team do the work for you We work for man
12. id filament aligns with homologous DNA Once aligned the 3 end becomes a primer for DNA replication E coli strain GBO5 dir harbours an arabinose inducible ETyA operon full length recE recT redy and recA at the ybcC locus The recombination window is therefore limited by the transient expression of the recombineering proteins Thus the risk of unwanted intra molecular rearrangement is minimized 4 Technical protocol 4 1 Generation of a vector backbone flanked by homology arms Oligonucleotide design i Choose 50 nucleotides at the 3 end of the fragment which you want to subclone Order an oligonucleotide with these 50 nucleotides at the 5 end At the 3 end of this sequence add the primer sequence for amplification of your plasmid backbone li Choose 50 nucleotides at the 5 end of the fragment which you want to subclone and transfer them into the reverse complement orientation Order an oligonucleotide with this sequence at the 5 end At the 3 end of this sequence add the primer sequence for amplification of your plasmid backbone PCR The oligonucleotides are suspended in dH20O at a final concentration of 10 pmol uL A standard PCR protocol is given below PCR reaction in 50 ul 38 5 ul dH20 5 0 ul 10 x PCR reaction buffer 2 0 ul 5 mM dNTP 1 0 ul upper oligonucleotide 1 0 ul lower oligonucleotide 1 0 ul PCR template 0 5 ul Taq polymerase 5 U ul e An annealing temperature of 57 62 C is optimal
13. leic Acids Res 27 1555 1557 1999 e Muyrers J P P Zhang Y Buchholz F and Stewart A F RecE RecT and Reda RedB initiate double stranded break repair by specifically interacting with their respective partners Genes Dev 14 1971 1982 2000 e Muyrers J P P Zhang Y Benes V Testa G Ansorge W and Stewart A F Point mutation of bacterial artificial chromosomes by ET recombination EMBO Reports 1 239 243 2000 e Zhang Y Muyrers J P P Testa G and Stewart A F DNA cloning by homologous recombination in Escherichia coli Nature Biotech 18 1314 1317 2000 e Muyrers J P P Zhang Y and Stewart A F Recombinogenic engineering new options for cloning and manipulating DNA Trends in Bioch Sci 26 325 31 2001 e Fu J Bian X Hu S Wang H Huang F Seibert P M Plaza A Xia L Muller R Stewart A F and Zhang Y Full length RecE enhances linear linear homologous recombination and facilitates direct cloning for bioprospecting Nature biotechnology 30 440 448 2012 Gene Bridges Direct cloning proficient E coli Strain GBO5 dir Version 1 1 May 2014 11 6 2 Patents Red ET recombination is covered by one or several of the following patents patent applications and related ones e Stewart A F Zhang Y and Buchholz F 1998 Novel DNA cloning method relying on the E coli RecE RecT recombination system European Patent No 1034260 United States Patent No 6509156 e
14. ration the cells are plated on the appropriate concentration of antibiotics depending on the copy number of the plasmid or BAC Similar number of colonies on both plates the induced and the uninduced one A similar number of colonies on both plates induced and uninduced one indicates that there are still traces of the circular or supercoiled plasmid used for preparing the linear plasmid backbone left in the sample Since the transformation efficiency of linear fragments is 10 fold less than that of circular DNA molecules you may obtain a background even if no traces were visible on an agarose gel If the linear DNA fragment was obtained by restriction digestion use less DNA and gel purify the fragment If the linear fragment was obtained by PCR set up a Dpnl digest for your PCR product and gel purify it at the end 10 Gene Bridges Direct cloning proficient E coli Strain GBO5 dir Version 1 1 May 2014 6 References and Patents 6 1 References e Zhang Y Buchholz F Muyrers J P P and Stewart A F A new logic for DNA engineering using recombination in Escherichia coli Nature Genetics 20 123 128 1998 e Angrand P O Daigle N van der Hoeven F Scholer H R and Stewart A F Simplified generation of targeting constructs using ET recombination Nucleic Acids Res 27 e16 1999 e Muyrers J P P Zhang Y Testa G and Stewart A F Rapid modification of bacterial artificial chromosomes by ET recombination Nuc
15. rotocols maps and sequences Please store tube 1 at 80 C tubes 2 and 3 at 20 C Gene Bridges Direct cloning proficient E coli Strain GBO5 dir Version 1 1 May 2014 3 2 Experimental Outline Linear DNA fragment ColE1 ori apR Linear plasmid backbone 1 Electroporation into GBOS dir cells Zi J RecE RecT Figure 1 Direct cloning workflow 1 Transform E coli GBO5 dir cells with a mixture of linear insert DNA and linear plasmid backbone For your convenience a linear plasmid backbone and a control insert kanamycin resistance marker gene sharing 50bp of terminal homology arms are provided for a control experiment Red ET Recombination step The expression of recE and recT genes mediating Red ET recombination is induced by the addition of L arabinose After induction the cells are prepared for electroporation and both linear fragments are electroporated Only colonies with circularized plasmid backbone will survive selection for ampicillin resistance Subsequent DNA mini preparation is used to confirm the successful integration of the DNA fragment Clones obtained from the control experiment can easily be analyzed for the correct insertion of the control fragment by a growth control test Only colonies carrying successfully modified plasmids will survive kanamycin plus ampicillin selection on the agar plates Gene Bridges Direct cloning proficient E coli Strain GBO5 dir Version 1 1 May 2014 3 How Re
16. ther products available from Gene Bridges u nna00000000nnnnnnnnnnnn nn nnnnnnnnnnnnnnnnnnnnn nn 12 9 DNA Engineering Services Available from Gene Bridges 2 n22aann0000nonnnnnnnnnnnn 143 Please read The products listed in this manual are for research purposes only They are not designed for diagnostic or therapeutic use in humans animals or plants Success depends on following the protocols exactly as they are described Do read the trouble shooting guide before beginning your experiments Red ET Recombination is the intellectual property of Gene Bridges GmbH Safety Some chemical reagents used with this system are dangerous if handled carelessly Take care when using chemical reagents such as isopropanol and ethidium bromide and electrical apparatus high voltage power supplies gel electrophoresis and electroporation apparatus Follow the manufacturers safety recommendations 2 Gene Bridges Direct cloning proficient E coli Strain GBO5 dir Version 1 1 May 2014 1 Direct cloning proficient E coli Strain GB05 dir Introduction Red ET recombination relies on homologous recombination in vivo in E coli and allows a wide range of modifications on DNA molecules of any size and at any chosen position Homologous recombination is the exchange of genetic material between two DNA molecules in a precise specific and accurate manner Homologous recombination occurs through homology regions which are stret
17. y commercial and research organizations across the world to provide DNA modifications in low or high copy plasmids cosmids BACs and the E coli chromosome Contact our DNA Engineering Service by email at contact genebridges com for details and prices 14 Gene Bridges Direct cloning proficient E coli Strain GBO5 dir Version 1 1 May 2014 This page intentionally left blank Gene Bridges Direct cloning proficient E coli Strain GBO5 dir Version 1 1 May 2014 Gene Bridges GmbH Commercial Centre Im Neuenheimer Feld 584 69120 Heidelberg Tel 49 0 6221 13 70 811 Fax 49 0 6221 13 70 829 contact genebridges com www genebridges com AAN
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