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Quick & Easy E. coli Gene Deletion Kit
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1. icnnical Protocol ill iF i nm ion t na ecomb 1 m Version 2 0 January 2007 CONTENTS 1 Quick and Easy E coli Gene Deletion Kit uerrssnnsennnnnnnnennnnnnnennnnnnnnnnnnnnnnnnnnnnnnnnnnnn nennen 2 2 Experimental OUNAE iere rennen 5 3 How Red ET Recombination works eese enne nnn nnn nennen nnn nnn nn nennen 7 4 Oligonucleotide Design for Red ET Recombination een 9 5 Media for antibiotic selection uurs rssunsenrnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnsnnnnnnnnnnnnn nan ERA nnn nnns 11 6 Technical protocol 1er ere rtt e cvectea steve core Fea dran ete ck nana e oua naso ka eroe 12 6 1 Generating a functional cassette flanked by homology arms lt lt lt 12 6 2 Transformation with Red ET expression plasmid pRedET eee 13 6 3 Disruption of a chromosomal DNA fragment by the FRT flanked PGK gb2 neo cassette 14 6 4 Verification of successfully modified genome by PCR analysis sess 16 6 5 Removal of the selection marker by FLP FLPe expression optional 17 6 6 Maps and Sequences ut ie pula te tuno iiie 20 7 Troubleshooting iere eie etenim la eid iene ard 24 8 References and Patents o ieicccecsieccedesscceccesseccenecnicecedesncue czegsecinevesiuecedesncieczeastcceenagsuededusssuedevacens 27 8 1 References een 27
2. High Red ET efficiency plus convenient removal of the Red ET Recombination protein expression plasmid pRedET after recombination Red ET Recombination protein expression plasmid pRedET Any E coli strain can be made Red ET proficient by transformation with this plasmid FRT or loxP flanked Kanamycin Neomycin resistance template FRT PGK gb2 neo FRT or loxP PGK gb2 neo loxP to be used for your own experiments Expression plasmid for FLPe or Cre site specific recombinase in E coli cells Positive controls to introduce a single FRT site into a 15 kb high copy plasmid Detailed protocols descriptions of plasmids maps and sequences Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 34 K007 Quick and Easy RNAi Rescue Kit Description Rescue the RNAi phenotype with a gene resistant to RNAi degradation ex mouse BACs for human cells and vice versa 1 BACs include promoters introns exons regulatory regions and therefore express the gene at physiological levels and with different splice variants This kit is designed as a quick and easy solution for the rescue of an RNAi phenotype using BACs for transient and stable transfections All components you need for the generation of the RNAi rescue construct included except for the BAC 1 Red ET recombination allows the Chloramphenicol resistance gene found on all BAC backbones pBeloBAC pBAC3 6 pTARBAC to be replaced by the SNAP26
3. and Muyrers J P P 1999 Methods and compositions for directed cloning and subcloning using homologous recombination United States Patent No 6 355 412 issued on 12 of March 2002 T Youming Zhang A Francis Stewart and Joep P P Muijrers 2001 Improved RecT or RecET cloning and subcloning method European Patent Application No 01 117 529 6 Stewart A F Zhang Y and Muyrers J P P 2001 Recombination method European Patent Application No 0103276 2 These patents and patent applications are owned by Gene Bridges or owned by the EMBL and exclusively licensed to Gene Bridges 29 Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 9 Purchaser Notification Warranty This product is the subject of European Patent No 1034260 issued on 12 of March 2003 or PCT EP98 07945 and United States Patent No 6 355 412 issued on 12 of March 2002 The purchase of this product conveys to the purchaser the non transferable right to use this product for research purposes only The purchaser can not 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 informatio
4. 10 sec 98 C 30 sec 55 C 90 sec 72 C final elongation step 10 min 72 C Check a 5 ul aliquot of the PCR product on a gel to ensure the PCR was successful The size of the PCR product for the FRT PGK gb2 neo FRT cassette is 1737 bp Purify the PCR product either by running the whole PCR sample on an agarose gel and subsequent gel extraction or directly by Spin Column e g Min Elute Gel Extraction Kit Qiagen Adjust the DNA concentration to 100 400 ng ul Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 12 6 2 Transformation with Red ET expression plasmid pRedET Before starting with the experiment please streak out your E coli culture on LB plates to obtain singly colonies Day 1 1 Set up an overnight culture Pick one or two colonies and inoculate them in microfuge tubes containing 1 0 ml LB medium Puncture a hole in the lid for air Incubate at 37 C overnight with shaking Day 2 Before starting Chill ddH2O or 10 glycerol on ice for at least 2 h Chill electroporation cuvettes 1 mm gap 1 Cool benchtop centrifuge to 2 C 1 Set up one or two microfuge tubes containing fresh 1 4 ml LB medium and inoculate with 30 ul of fresh overnight culture 2 Culture for 2 3 h at 37 C shaking at 1000 rpm 3 Prepare the cells for electroporation Centrifuge for 30 sec at 11 000 rpm in a cooled microfuge benchtop centrifuge at 2 C Discard the superna
5. C overnight or for at least 15 h Protect the plates from light by wrapping them up because tetracycline is sensitive to light Make sure the cells stay at 30 C otherwise the plasmid will be lost 6 3 Disruption of a chromosomal DNA fragment by the FRT flanked PGK gb2 neo cassette In the next step prepare electro competent cells that contain the Red ET expression plasmid shortly after inducing the expression of the recombination proteins In advance prepare the linear DNA fragment the FRT PGK gb2 neo FRT cassette with homology arms that you will use to replace the DNA fragment on the chromosome Use tube 4 FRT PGK gb2 neo FRT PCR product to perform the control experiment in parallel Day 3 T To start overnight cultures pick one colony from the plate you obtained in 6 2 step 8 and inoculate one microfuge tube containing 1 0 ml LB medium plus either tetracycline 3 pg ml or ampicillin 50 pg ml depending on which pRedET plasmid you used in your experiment Also pick one colony from the control plate and inoculate one microfuge tube containing 1 0 ml LB medium plus tetracycline 3 ug ml each Puncture a hole in the lid of the tubes for air Incubate the cultures while shaking at 30 C over night overnight Day 4 Before starting Chill ddH2O or 10 glycerol on ice for at least 2 h Chill electroporation cuvettes 1 mm gap 1 Cool benchtop centrifuge to 2 C 2 The next day set up 4 lid punctured microf
6. A103 into the cells and subseguent expression of FLP site specific recombinase Prepare electro competent cells from a clone harboring the FRT flanked kanamycin neomycin cassette on the chromosome and electroporate the FLP expression plasmid into the cells 706 FLP carries a pSC101 origin of replication The plasmids will maintain low copy and replicate at 30 C They will not propagate and will get lost when incubated at 37 C The expression of the FL P recombinase is driven by the thermo sensitive promoter cl578 lpr promoter Therefore the expression is repressed at 30 C and induced at 37 C The plasmid carries a tetracycline resistance gene Day 5 1 Set_up an overnight culture Pick one or two colonies carrying the FRT flanked kanamycin neomycin cassette and inoculate them in microfuge tubes containing 1 0 ml LB medium and 50ug ml kanamycin Puncture a hole in the lid for air Incubate at 37 C overnight with shaking 17 Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 Day 6 Before starting Chill ddH2O or 10 glycerol on ice for at least 2 h Chill electroporation cuvettes 1 mm gap 1 Cool benchtop centrifuge to 2 C 2 Set up a microfuge tube containing fresh 1 4 ml LB medium conditioned with 50ug mi kanamycin and inoculate with 30 ul of fresh overnight culture 3 Culture for 2 3 h at 37 C shaking at 1000 rpm 4 Prepare the cells for electroporation Centrifuge f
7. IGG ATC GAT TIC GIT 112 CCA GGT GAR AAT GCC GAA ACG CIE ATT GAA AAG TAC AAC GCT CAG TTG PCR primer 5 homology sm EHR 166 GCA AAA CIC GAC ACC ACT AAA GGC GIG CIE TIT CIC SIT GAT ACA TGG zoe GGA GET ASC CCS TIC AAT GCT SCC ASC CGC AIT GIC GI aarraacceicactasa Netl 817 C TGCAGC AGCACGICTI GACARTTART CATOGGCATA GIRTATOGGC ATAGIATAAT ACGACAAGGT GAGGAACT A 854 ACC ETG GGA TCG Glo ETT GSA C GAT Ga TTG CET SCA GT TCT ug SCS GT TOS GIG u iSc 17M G S A E Qa D G6 L H A G5 PA AW V E R 960 CTA TIC GOC TAT GAC TOG GCA CAR CMG ACS ATC CSC TOC ICT Gat GCC GOC GIG TIT C96 CIG TCA a UE GN O MA G rf ke S D A A VER LS 1026 309 CIE 5549 CGC 000 GIT CTT TIT GTC AE ACT SAT CTO TOC S5T SO TG AIT SKA CIC CM ac sA OG RP V L FV K TDLS GA LN EL GD 1092 GAG GOA GOS CSG CTA TCG TOG CTG GOC NOG ACG GSC GIT OCT TeC GCA GET STG CTC GAC S GIC MEA EEE hd FF 87V PUE AA Y Oo ey Y 1158 ACT GRA GOG GEA AGO GAC TGG CIS CTA TIG GSC GAA STG CCE S60 CAG GAT CIC CTG TCA NCT CAC aT E A G R DW L L L G E VP G GOD L L S S H 1224 CIT SET OCT SE GAS MAA Gta TEC ATC Ate SCY cit SCA Ate Cog COG CG CAT ACS OTT Git CCS ort A A E KV S 1 MA DA RR RO H T E P 1280 GT MCT TEC CIA TIC SAC CAT CEA SCS AAA CAT CSC AIC gap EA GC CET ACT COG KIS SA GC wea T C PF DH Q AK HRI ER A R TR NME A i256 SET CIT GRC SAT CAS GAT GAY CIS CAC SAA CAS CAT CAE GOS CIC X6 COA SOC GK CIE IC XC 34 6 L VD GD BA BE E H Q S8 L A FP A E L F A 1422 253 CIC 443 Ge CS ETG CCT CAT Gee S25 84T CIC S
8. be lost Day 7 10 Pick a single colony and grow the cells in 1 ml of LB medium without antibiotics at 30 C for 2 3 h 11 Change the temperature to 37 C and incubate overnight FLP FLPe protein will be expressed at this temperature and the FRT sites will be recombined At the same time the expression plasmid cannot replicate any more and will get lost Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 18 Day 8 12 Take a loop and streak a sample of the culture on a fresh LB agar plate To obtain single colonies one drop is enough Incubate overnight at 37 C The next day analyze twelve single colonies by PCR for the successful removal of the selection marker Alternatively you can streak the colonies on two different LB agar plates in parallel one conditioned with 15ug ml kanamycin the other one without addition of antibiotics Clones growing only on LB agar plates without kanamycin but no longer on kanamycin conditioned LB agar plates have successfully removed the selection marker by FLP recombination Figure 7 Marker test Single Colonies were streaked out on LB plates left and LB kanamycin plates right in parallel The four clones growing on the kanamycin plate still contain the selection marker cassette while all other clones lost the selection maker by FLP recombination 19 Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 6 6 Maps and Seque
9. 5 end At the 3 end of this oligo include the PCR primer sequence for amplification of the FRT PGK gb2 neo FRT cassette given in italics below Upper oligonucleotide 5 N so AATTAACCCTCACTAAAGGGCG 3 Il Choose 50 nucleotides N so directly adjacent downstream 3 to the intended insertion site and transfer them into the reverse complement orientation Order an oligonucleotide with this sequence at the 5 end At the 3 end of this oligo include the 3 PCR primer sequence for the FRT PGK gb2 neo FRT cassette given in italics below Lower oligonucleotide 5 N so TAATACGACTCACTATAGGGCTC 3 If desired include restriction sites or other short sequences in the ordered oligo s between the 5 homology regions and the 3 PCR primer sequences Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 10 5 Media for antibiotic selection All antibiotics are available from Sigma Stock solutions should be stored at 20 C For selective LB medium the antibiotic is dissolved in LB medium to the indicated working concentration 1 Chloramphenicol stock solution c 30 mg ml dissolved in ethanol Working concentration 50 ng ml Ampicillin stock solution c 100 mg ml dissolved in 50 ethanol Working concentration 50 ng ml Tetracycline stock solution c 10 mg ml dissolved in 75 ethanol Working concentration for pRedET is 3 ng ml Tetracycline is light sensitive Kanamycin stoc
10. 8 2 PUO eE E sn R E O R RERE 29 9 Purchaser Notification Warranty uuensesnssnrennnnnennnnnnnnnnnnnnnnnnnnnnnnennnnnnnnnnnnnnnnnnrnsnnnenrnsn nennen 30 10 Other products available from Gene Bridges serene nnnm 31 11 DNA Engineering Services Available from Gene Bridges eese 37 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 carelessiy 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 1 Quick and Easy E coli Gene Deletion Kit Introduction Targeted disruption of genes on the E coli chromosome Metabolic engineering to design and construct microorganisms suitable for the production of industrial products like ethanol or aromatic amino acids reguires the disruption of specific genes on the bacterial chromosome Regulatory circuits the uptake of carbon and amino acids the glycolytic and pe
11. ATTGTTATAGGCACACATGGTTGGGCTGCAGAGCAGTTGCTTAARACGGCAGAA ATG CIG TIA GGC GAG CAG GAA AAC GTC GGC TGG ATC GAT TIC GIT CCA 1bMst Leu Leu Giy Giu uz GAR AAT GCC GAA ACG 18b Giu Asn Als Glu Thr PCR primer 1 ies GAC ACC ACT AAA GGC Gin CIG Lev Glu Asn ATT GAA Ile Glu 220 TIC AAT GCT SCC AGC 52PPhe Asn Als Als Ser 271 SCA GGT GIT AAC AIT amp b Ais Gly Vai Asn lle 322 GAC CCA AGC TIT GAT sehAcp Fro Ser Phe Asp 273 GGC GIG AAA GCA CIE 102 Gly Val Lys Als Leu 424 GCI GCC GCA GCA CCA izobAis Als Als Als Pro 415 AAC GAC TAC AIG GIT 130bAsn Asp Tyr Met Val 526 GGI CAG GIC GEC ACC 154 Gly Gin Vai Als Thr 577 GIT GIT ACT GAT GAA 1710 Vai Val Ser Asp Giu ez CAG GIT GCA CCT CCG 182b Gin Val Ala Pro Pro ers ATT CGC GTC TAC AAC zosh ie Arg Val Tyr Asn Tae TIT ACC AAC CCA ACA zzzbhPhe Thr Asn Pro Thr 73831 ACC TCI GIT AAC GIC 239 Thr Ser Val Asn Val aaz AAT AAC GCG GIT TCG 2560 Asn Asn Ais Val Ser 883 AAT GCG CGC GGT ATT 273 Asn Ala Arg Giy lie 834 CIE AAA ATG ATG GAT 299b Leu Lys Met Met Asp Arg GIG Val ccc Gly RAC Asn GAT Asp GGI Giy GIT Val GAG Giu Val Giy Trp Ile Asp Phe Val Pro AAG TAC AAC GCT CAG TIG GCA AAA Lys Tyr Asn Als Gin Lev Ala Lys 5 homslogy arm Seg CIC SIT GAT ACA IGS SGA GET ASC Leu Val Asp Thr Trp Gly Gly Ser Insertion ste of he anamyon SEE marea STC GAC AAR GAG CAT TAT GAA GTC Vai Asp Lys Glu His Tyr Giu Val gt homelogy arm SObp GTG GAA ACG TTA ATG GCC CGI GAT
12. GCT TTGCTCCTTC GCTTTCTGGG CTCAGAGGCT 422 GGGAAGGGGT GGGTCCGGGG GCGGGCTCAG GGGCGGGCTC AGGGGOGGGG CGGGCGCCCG AAGGTCCTCC 492 GGAGGCCCGG CATTCTGCAC GCTTCAAAAG CGCACGTCTG COGCGCTGTT CTCCTCTTCC TCATCTCOGG 562 GCCTTTCGAC C TGCAGC AGCACGTGTT GACAATTAAT CATCGGCATA GTATATCGGC ATAGTATAAT 629 ACGACAAGGT GAGGAACTAA ACC ATG GGA TOG GCC ATT GAA CAA GAT GGA TTO CAC GCA GIT ib Met Gly Ser Ala Ile Glu Gin Asp Gly Leu His Ala Gly 691 TCT COG GOC GCT TGS GIG GAG AGG CTA TTC GGC TAT GAC TGG GCA CAA CAG AOG ATC 14b Ser Pro Ala Ala Trp Val Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gin Gin Thr Ile 748 GGC TGC TCT GAT GCC GCC GTG TTC OGG CTG TCA GOS CAS GGG GC cos GIT CTT TTT 33b Gly Os Ser Asp Ala Ala Val Phe Arg Leu Ser Ala Gin Gly Arg Pro Val Leu Phe 805 GTC AAG ACC GAC CTG TCC GGT GCC CTG AAT GAA CTG CAG GAC GAG GCA GCG COS CTA 52 Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gin Asp Glu Ala Ala Arg Leu 862 TOG TG3 CTG GCC ACG ACG GAC GIT CCT TGC GCA GCT GTG CTC GAC GIT GTC ACT GAA 71 gt Ser Trp Leu Ala Thr Thr Gly Val Pro Os Ala Ala Val Leu Asp Val Val Thr Glu 919 GOG GGA AGG GAC TGS CTG CTA TIG GGC GAA GTG COG GGG CAG GAT CTC CTG TCA TCT 90h Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu Val Pro Gly Gin Asp Leu Leu Ser Ser 976 CAC CTT GET CCT GCC GAG AAA GTA TOC ATC ATG GCT GAT GCA ATG COGG OGG CTS CAT 109b His Leu Ala Pro Ala Glu Lys Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His 1033 AOS CIT GAT COG GCT ACC TGC CCA TTC GAC CAC CAA GOG AAA CAT CGC ATC GAG OGA 128b Thr Leu Asp P
13. M kanamycin neomycin cassette within 1 week The SNAP cassette is easily detected by TMR Star dye Cells carrying the gene variant resistant to RNAi degradation can be identified within 30min Contents Red ET Recombination protein expression plasmid pRedET Any E coli strain can be made Red ET proficient by transformation with this plasmid Ready to go SNAP26M kanamycin neomycin cassette flanked by homology arms E coli strain HS996 already carrying the pRed ET plasmid and an unmodified BAC clone for the control experiment Positive control BAC containing the SNAP26M cassette Primers necessary to check for correct replacement of Chloramphenicol resistance gene with SNAP26M cassette Reagents needed for SNAP detection including the SNAP tag substrate TMR Star Detailed protocols descriptions of plasmids maps and sequences 35 Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 Additional functional cassettes A001 Pro and Eukaryotic Neomycin Selection Cassette PGK gb2 neo A002 FRT flanked Pro and Eukaryotic Neomycin Selection Cassette FRT PGK gb2 neo FRT A003 loxP flanked Pro and Eukaryotic Neomycin Selection Cassette loxP PGK gb2 neo loxP A004 FRT flanked Pro and Eukaryotic Neomycin Selection Cassette plus loxP site FRT PGK gb2 neo FRT loxP A005 FRT flanked Pro and Eukaryotic Neomycin Selection Cassette plus loxP site 2 version loxP FRT PGK
14. Vai Glu Thr Leu Met Als Arg Asp GCA CIG GCA GIA GAA ACA GGC CGT Ais Leu Aia Val Glu Thr Gly Arg CCG GIT GAA AAA GCC GCG CCA GCA Pro Val Giu Lys Ala Als Pro Als PCR primer 4 CCA ACT CCG GCA AAA CCA ATG GGG Pro Thr Pro Als Lys Pro Met Gly GCG CGT ATC GAC GAC CGT CIG AIT Ais Arg ie Asp Asp Arg Leu lie TGG ACC AAA GAA ACC AAT GIC ICC CGI AIT Trp Thr GCI GCG Alz Als GTA ACA Vai Thr CCG ARA Pro Lys GTA GAG Val Glu GGT AIG Gly Met GAT GAA Asp Glu CIE GAA Leu Giu Lys Glu Thr Asn Vai Ser Arg lle GAT ACC GTT CST AAG ACA CTG CIC Asp Thr Val Arg Lys Thr Leu Leu GCA CAC GIA GIT GAT GIT GCC AAA Ala His Val Val Asp Val Ala Lys TAT GCT GGC GAA CGC GTA ATG CIG Tyr Ala Gly Giu Arg Val Met Leu CGI CIC GIT GAA GGC GGC GTG AAA Arg Leu Va Glu Gly Gly Vai Lys GCA TIC CGI CAG GET AAA ACC CAG Ais Phe Arg Gin Gly Lys Thr Gin AAA GAT ATC GAS GCG TIC AAG AAA Lys Asp Ile Glu Ala Phe Lys Lys GIC CGT AAG GIT TCC ACC GAT CCG Val Arg Lys Val Ser Thr Asp Pro CIG ATC AGC AAA ATC GAT AAG TAA Lev Ile Ser Lys Ile Asp Lys Figure 11 manX gene locus on the E coli chromosome Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 GGT Sly CIC Leu ccs Pro Glu ccc Pro CCA Pro CAC His ATT lle ACC Thr ATS Met TTA Leu ATC Ite GTG Vai CTS Leu ARR Lys 22 1 GTGACCATTGCTATTGTTATAGGCACACATGGTTGGGCTGCAGAGCAGTTGCTTAAAACGGCA e GAA ATG CIG TTA GGC GAG CAG GAA AAC GTC GGC
15. anabe H Determination of the InvE binding site required for expression of IpaB of Shigella sonnei virulence plasmid involvement of a parB boxA like sequence J Bacteriol 185 5158 5165 2003 1 Uzzau S Figueroa Bossi N Rubino S and Bossi L Epitome tagging of chromosomal genes in Salmonella PNAS 98 15264 15269 2001 q Yu D Ellis H M Lee E C Jenkins N A Copeland N G and Court D L An efficient recombination system for chromosome engineering in Escherichia coli PNAS 97 5978 5983 2000 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 1 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 Zhang Y Muyrers P P J Rientjes J and Stewart A F Phage annealing proteins promote oligonucleotide directed mutagenesis in Escherichia coli and mouse ES cells BMC Molecular Biology 4 1 14 2003 Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 28 8 2 Patents Red ET recombination is covered by one or several of the following patents and patent applications T Stewart A F Zhang Y and Buchholz F 1998 Novel DNA cloning method European Patent No 1034260 issued on 12 of March 2003 United States Patent No 6 509 156 Stewart A F Zhang Y
16. as introducing short sequences e g point mutations loxP sites restriction sites etc insertion or deletion of non selectable marker genes fragment exchange without leaving a selection marker or any unwanted sequences The included counter selection cassette pRpsL neo is based on Streptomycin selection which shows a much higher efficiency than pSacB neo or comparable systems This kit can also be used to work on bacterial chromosomes and common ColE1 origin plasmids High Red ET efficiency plus convenient removal of the Red ET Recombination protein expression plasmid pRedET after recombination Red ET Recombination protein expression plasmid pRedET Any E coli strain can be made Red ET proficient by transformation with this plasmid BAC host E coli strain HS996 already carrying the Red ET plasmid pRpsL neomycin template to be used for your own experiments Positive controls to introduce a point mutation in a 150 kb BAC Detailed protocols descriptions of plasmids maps and sequences Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 32 K003 BAC Subcloning Kit Description This kit is optimized for subcloning of DNA fragments from BACs and cosmids No restriction sites necessary Fragments up to 20 kb can be subcloned High Red ET efficiency plus convenient removal of the Red ET Recombination protein expression plasmid pRedET after recombination Contents Red ET Recom
17. between a linear DNA molecule e g a PCR product and circular DNA plasmid BAC or E coli chromosome This method was used to disrupt the endogenous acZ gene of E coli strain JC9604 Zhang et al 1998 One year later the system was extended by the same group in replacing recE and recT by their respective functional counterparts of phage lambda redU and redb Muyrers et al 1999 Since the year 2000 the system which is protected by several international patents from Gene Bridges has been used by other academic groups to disrupt several chromosomal genes in E coli e g Datsenko and Wanner 2000 Yu et al 2000 The Auick and Easy E coli Gene Deletion Kit from Gene Bridges provides an optimized system for the disruption of genes on the E coli chromosome in a seguence precise manner attainable within one week The use of a FRT flanked resistance cassette for the disruption or replacement of the targeted gene allows the subsequent removal of the selection marker by a FL P recombinase step optional if reguired Two Red ET expression plasmids are provided with the kit mediating either tetracycline or ampicillin resistance The choice of the expression plasmid allows the user to perform Red ET recombination even if one selection marker is already present in the E coli strain Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 2 The control experiment included in the kit demonstrates the easy and precise
18. bination 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 encoded Gam protein which inhibits the RecBCD exonuclease activity of E coli Double stranded break 3 5 l RecE 2 or Reda exonuclease 5 3 3 l Roc m is A v eo orRedB ingle stran binding proteins 5 3 3 Joint molecule formation DNA replication Figure 2 Mechanism of Red ET Recombination 7 Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 Double stranded break repair DSBR is initiated by the recombinase protein pairs RecE RecT or Reda Redb First Reda or RecE digests one strand of the DNA from the DSB leaving the other strand as a 3 ended single stranded DNA overhang Then Redb or RecT binds and coats the single strand The protein nucleic acid filament aligns with homologous DNA Once aligned the 3 end becomes a primer for DNA replication The recombination proteins can be expressed from a plasmid Figure 5 and are therefore transferable to any E coli strain pRedET Figure 5 carries the aphage redgoa operon expressed under the control of the arabinose inducible pBAD promoter Guzman et al 1995 and confers Tetracycline resistance The pBAD promoter is both positively and negatively regulated by the product of the araC gene Schleif 1992 AraC
19. bination protein expression plasmid pRedET Any E coli strain can be made Red ET proficient by transformation with this plasmid Linear vector carrying a ColE1 origin of replication plus Ampicillin resistance gene to be used for the subcloning experiment Positive controls to subclone a 15 kb fragment from a control BAC into the vector delivered with the kit Detailed protocols descriptions of plasmids maps and sequences 33 Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 K004 Quick and Easy Conditional Knockout Kit FRT FLPe and K005 Quick and Easy Conditional Knockout Kit loxP Cre Description Contents This kit is designed to integrate FRT or loxP sites into large vectors at any position within 2 weeks Single FRT or loxP sites are inserted by Red ET recombination of FRT or loxP flanked functional cassettes into any designated locus with subseguent removal of the selection marker by FLPe or Cre recombinases Conditional targeting constructs can be generated either by a repetitive insertion of the functional cassette supplied with the kit or by combination with other functional cassettes offered by Gene Bridges The functional cassette supplied with the kit FRT PGK gb2 neo FRT or loxP PGK gb2 neo loxP combines a prokaryotic promoter gb2 for expression of Kanamycin resistance in E coli with a eukaryotic promoter PGK for expression of Neomycin resistance in mammalian cells
20. by about 80 during four generations of bacterial cell growth at 42 C After return of the cultures to 30 C approximately the same number of generations of bacterial cell growth is required for the copy number of the plasmid to return to the level observed before Miller Ingmer and Cohen 1995 Since the plasmid is based on oriR101 it can be propagated in E coli together with most ColE1 derived plasmids Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 8 4 Oligonucleotide Design for Red ET Recombination To target the chromosome at the site s of choice it is necessary to attach incorporate short homology regions into the functional cassette carrying the selectable marker Sm This is most conveniently done by ordering two oligonucleotides for use in PCR amplification Figure 3 Each oligonucleotide consists of two or if desired three parts 1 Required Part A A for the second oligonucleotide is the homology region shared by the target molecule and the linear molecule The homology regions are the 50 bp directly adjacent to either side of the insertion site The exact sequences of the homology regions can be chosen freely depending on the position on the target molecule to be modified 2 Optional Part B B gfor the second oligonucleotide This part of the oligonucleotide allows the incorporation of useful sequences such as restriction sites If the introduction of such operational se
21. d other secreted proteins in infection Infection and Immunity 72 2288 2302 2004 Murphy K C and Campellone K G Lambda Red mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E coli BMC Molecular Biology 4 1 12 2003 Muyrers J P P Zhang Y Testa G Stewart A F Rapid modification of bacterial artificial chromosomes by ET recombination Nucleic Acids Res 27 1555 1557 1999 1 Muyrers J P P Zhang Y Buchholz F 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 Muyrers J P P Zhang Y Stewart A F ET cloning Think recombination first Genetic Engineering Principles and Methods Ed J K Setlow 22 77 98 Kluwer Academic Plenum Publishers NY 2000 1 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 27 Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 Schleif R S DNA Looping Annu Rev Biochem 61 199 223 1992 Sapriel G Wandersman C and Delepelaire P The secB chaperone is bifunctional in Serratia marcescens secB is involved in the sec pathway and required for hasA secretion by the abc transporter J Bacteriol 185 80 88 2003 Taniya T Mitobe J Nakayama S Mingshan A Okuda K and Wat
22. ed strains in very rare cases an elongation of the reaction time for the recombination from 70 min incubation of electroporation to up to four hours is necessary for successful recombination 25 Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 4 Problems with and after the electroporation cells are not competent enough to take up the linear DNA fragment Please make sure that the cells were kept on ice and that the water respectively 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 please make sure that there is no arching during the electroporation process please make sure that after electroporation the cells are plated on the appropriate concentration of antibiotics Similar number of colonies on both plates the induced and the uninduced one If you obtain a high number of colonies on both plates it indicates that there are still traces of the circular or supercoiled plasmid used to prepare the linear fragment left in the sample Since the transformation efficiency of linear fragments is 10 fold less than 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 ge
23. et repeats directly Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 24 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 PCR product could be wrong check it by restriction digest or sequencing could be degraded check an aliquot on an agarose gel could have incorrect homology arms Please double check the oligonucleotides used to generate the homology arms for quality and correctness If necessary verify the sequence by sequencing of the PCR product may not be enough increase the amount of PCR product from approximately 200 ng up to 500 ng Please take into consideration that you may also increase non specific background 2 The region on the chromosome the sequence which should be recognized by the homology arm is different or absent check for the presence by amplifying the region by PCR and sequence the PCR product if necessary 3 The Red ET reaction did not take place because there was no expression plasmid present in the cells e g the cells were grown at 37 C instead of 30 C check for tet resistance no or the wrong type of arabinose was used for induction please make sure you use L arabinose some strains e g JM109 DHb5alpha are less efficient in Red ET Recombination than others DH10B HS996 GeneHogs or TOP10 are our preferr
24. experimental design before starting High quality oligos yield highest recombination efficiencies Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 4 2 Experimental Outline 1 step Generation of a PCR product from the functional cassette flanked with homology arms oligo up N PCR _ 000000000 E gt o 0000 MM oligo down FRT PGK gb2 neo FRT functional cassette template flanked with homology arms FRT sites 2 step Transformation of pRedET into the E coli host 3 step Induction of the Red ET recombination genes and subsequent transformation of the linear PCR product into the E coli host induction transformation Red ET target locus Figure 1 Flowchart of the experimental outline for the targeted disruption of genes on the E coli chromosome 5 Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 5 step Transformation of FLP expression plasmid into the E coli host optional target locus E coli chromosome FLP Figure 1 continued Flowchart of the experimental outline for the targeted disruption of genes on the E coli chromosome In the first step 50bp long homology arms corresponding to the seguences flanking the insertion site on the chromosome are added to the functional cassette by PCR In parallel the E coli strain which will be modified is transformed with the expression plasmid pRedET step 2 The ex
25. flanked kanamycin neomycin resistance marker cassette They are supplied with the kit tubes 7 to 10 PCR primer 1 5 CACCACTAAAGGCGTGCTGT 3 PCR primer 2 5 CGAGACTAGTGAGACGTGCTAC 3 PCR primer 3 5 TATCAGGACATAGCGTTGGCTACC 3 PCR primer 4 5 TGGAGCCGCTTTTGGTGCT 3 7 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 Several 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 Please ask you local oligonucleotide supplier for the recommended purification Homologous recombination will also not take place if the targeted sequence on the chromosome is not determined 100 correctly for the homology region If you are trying to target a repeated sequence you may experience problems because the homology region at the end of the linear fragment can go to more than one site It is therefore best not to targ
26. fragment tube 9 and 10 Since for each pair one primer is located on the chromosome and the second one on the cassette only a correctly recombined fragment will yield the expected PCR product confirming the correct insertion of the FRT flanked kanamycin neomycin resistance cassette E coli ETEMA 40 9 41 0 Red ET recombination J pRedET plasmid FRT neo FRT cassette 1aib 23 4a gt gt lt gt many oe FRT flanked Flp recombination J 705 FLP plasmid t Figure 5 Confirmation of the correctly integrated FRT flanked kanamycin neomycin selection cassette Primers 1b 2 3 and 4a are supplied with the kit Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 16 M 1 2 3 4 5 M 67 8 9 10 BAND SIZE bp ng BAND 10 000 100 8 000 80 6 000 60 5 000 50 4 000 40 3 000 30 2 500 25 2 000 1 500 15 Figure 6 PCR primer combinations 3 4a lane 1 3 4b lane 2 1b 2 lane 3 and 1a 2 lane 4 confirm the correct insertion of the cassette by Red ET recombination After FLP recombination primer combination 1b 4a lane 5 amplifies the DNA fragment without the cassette M Hyperladder Bioline 6 5 Removal of the selection marker by FLP FLPe expression optional Since the kanamycin neomycin selection marker is flanked by FRT sites the cassette can be removed by transformation of a FLP expression plasmid e g 706 FLP Gene Bridges Cat No
27. gb2 neo FRT A006 FRT flanked Chloramphenicol Selection Cassette FRT cm FRT A007 loxP flanked Chloramphenicol Selection Cassette loxP cm loxP A008 FRT flanked Ampicillin Selection Cassette FRT amp FRT A009 loxP flanked Ampicillin Selection Cassette loxP amp loxP Additional strains and plasmids A A A A A A A A101 FLP Expression Strain 294 Flp A102 FLP Expression Plasmid 705 Flp cm resistance marker A103 FLP Expression Plasmid 706 Flp tet resistance marker A111 Cre Expression Strain 294 Cre 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 Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 36 11 DNA Engineering Services Available from Gene Bridges Instead of performing your own DNA manipulations let the Gene Bridges DNA Engineering Service do the work for you We work for many commercial and research organisations across the world to provide DNA modifications in low or high copy plasmids cosmids BACs and the E coli chromosome The available DNA modifications are Insertion of a selectable or non selectable marker cassette Deletion of sequences of any size ranging from 1 bp up to more than 100 kb with or without leaving a selectable marker Replacement of genes on the E coli chromosome Point mutations A Fusio
28. is a transcriptional regulator that forms a complex with L arabinose Arabinose binds to AraC and allows transcription to begin In the presence of glucose or the absence of arabinose transcription is blocked by the AraC dimer The plasmid carries the reda b ggenes of the phage together with the recA gene in a polycistronic operon under the control of an inducible promoter The recombination window is therefore limited by the transient expression of Red proteins Thus the risk of unwanted intra molecular rearrangement is minimized While constitutive expression of the red gene has a toxic effect in recA cells like DH10B or HS996 under some conditions thus limiting the efficiency of recombination tightly regulated expression of the gene together with simultaneous expression of the red and genes allows efficient homologous recombination between linear DNA fragments and plasmids resident in cells such as DH10B pRedET is a derivative of a thermo sensitive pSC101 replicon which is a low copy number plasmid dependent on oriR101 The RepA protein encoded by plasmid pSC101 is required for plasmid DNA replication and the partitioning of plasmids to daughter cells at division Miller Ingmer and Cohen 1995 Because the RepA protein is temperature sensitive Ts cells have to be cultured at 30 C to maintain the plasmid pSC101 derivatives are easily curable at 37 C to 43 C Experiments have shown that the copy number of the plasmid decreases
29. it GIG ACT CAT Ge CAST zo TST TTS m veR L K A RM PDG E DL VVT H G DA Ct P 1488 AAT ATC ATG GIS GRA ART GOC CSC TIT TCT Gea TIC ATC GAC TOT CSC COS CIE Gor GIG GOs GAC 189 N M V E NG R F S GF DC G6 R L GV A D PCR primer 3 20h R Y O D A L T RB 1 A E E L 6 G E WA D 1620 ot TIC CIC als CIT Tac SST Are QOO GET COC eat ECG cas cot ETC coc TIC MAY OSC CIT CIT MFR F L V L Y G A A P DS QR AF Y R t L 1686 SAS 235 TTC TIC TEA TARAGACCGACCAAGCGAC GIC TGA GASTTOCTTS 178 GOGAATTCSG TACCAATAAA AGAGCTTTAT TTICATGATC TGTGTETTGG TTTTTGICTG CGGCGCG GAAGTICCTATICT Xhal 1837 CTAGAAACTRIACGAACTTC C TOGAGCOCT TASTS STOGT TTA CGA CAA AGA GCA TIA TGA AGT Themology arm 60bp 1904 CAT TGC ASG CGT TAA CAT TCC AAT GCT CET GGA AAC GTI AAT GGC CCE i552 TGA TGA TGA CCC AAG CIT TGA IGA ACT GGT GGC ACT GGC AGT AGA AAC 2000 AGG CCG TGA AGG CGT GAA AGC ACT GAA AGC CAA ACC GGI TGA AAA AGC PCR primer 4 2048 CGC GCC AGC ACC CGC TGC CGC AGC ACC AAA AGC GGC ICC AAC ICC GGC zose AAA ACC AAT GGG GCC AAA CGA CTA CAT GGT TAT TGG CCI TGC GCG TAT 2144 CGA CGA CCG ICT GAT TCA CGG TCA GGT CGC CAC CCG CTG GAC CAA AGA zisz BAC CAA Figure 12 manX gene locus after insertion of the FRT flanked kanamycin neomycin selection marker cassette 23 Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 Oligonucleotides The four oligonucleotides labeled PCR primer 1 to PCR primer 4 are designed to verify the correct insertion of the FRT
30. k solution c 30 mg ml dissolved in ddH20 Working concentration 15 ng ml Selective LB plates are made by adding 15 g agar to 1 L LB medium After boiling cool to approx 50 C add the required antibiotics to yield the appropriate working concentrations and pour into petri dishes L arabinose stock solution Use 10 L arabinose Sigma A 3256 in ddH2O fresh or frozen in small aliquots at 20 C Use 50 ul stock solution per 1 4 ml LB for induction of recombination protein expression from pRedET Frozen aliquots should not undergo more than three freeze thaw cycles 11 Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 6 Technical protocol 6 1 Generating a functional cassette flanked by homology arms PCR The oligonucleotides are suspended in ddH2O at a final concentration of 10 uM We present as an example a standard PCR protocol for the use of Phusion DNA Polymerase Finnzyme However any other DNA Polymerase together with the corresponding PCR protocol according to the instructions of the manufacturer should yield satisfactory results PCR reaction in 50 ul 34 5 ul dH O 10 0 ul 5 x HF PCR reaction buffer 2 0 ul 5 mM dNTP 1 0 ul upper oligonucleotide 1 0 ul lower oligonucleotide 1 0 ul FRT PGK gb2 neo FRT PCR template tube 3 0 5 ul Phusion polymerase 5 U ul An annealing temperature of 57 62 C is optimal 1 PCR Profile Initial denaturation step 30 sec 98 C thirty cycles
31. knock out of a chromosomal gene in this case the major mannose transporter manXYZ of E coli strain HS996 using the provided FRT flanked kanamycin resistance cassette The recombination process is strictly controlled since the necessary genes are located on an expression plasmid carrying a temperature sensitive origin of replication and can therefore only be propagated at 30 C An increase of the temperature to 37 C results in a loss of the expression plasmid after recombination In addition the expression of the proteins is tightly controlled by an inducible promoter opening just a short time window for the recombination process There is proven evidence that Red ET recombination works not only in E coli but also in other microorganisms like Salmonella e g Uzzau et al 2001 Shigella Taniya et al 2003 Yersinia Derbise et al 2003 Serratia Sapriel Wandersman and Delepelaire 2003 and Citrobacter Mundy et al 2004 Remark The efficiency of Red ET recombination may vary between different E coli strains 3 Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 Contents of the kit T 2 d pRedET tet Red ET expression plasmid 20 ng ul 20 ul pRedET amp Red ET expression plasmid 20 ng ul 20 ul FRT PGK gb2 neo FRT template DNA PCR template plasmid DNA for generating a FRT flanked PGK gb2 neo cassette 50 ng ul 20 ul FRT PGK gb2 neo FRT PCR product PGK gb2 neo cassette f
32. l purify the fragment If the linear cassette was obtained by PCR set up a Dpnl digest for your PCR product and gel purify it at the end If you obtain a very low number of colonies on both plates it indicates that the overall efficiency of Red ET Recombination is very low In this case please check all parameters mentioned in the section entitled no colonies after Red ET Recombination High number of false positive colonies on the induced plates If you obtain a high number of colonies on the induced plates for which you cannot confirm the correct location of the insertion please double check the sequences you used as homology arms for the recombination by BLAST analyses against the E coli genome Avoid highly conserved regions and repetitive sequence stretches since otherwise the primer may guide the cassette to a wrong location Modification of E coli strains Modification of the E coli genome We observed that the efficiency of Red ET recombination varies between different E coli strains Although the overall number of colonies was less for strain W3110 in all experiments performed the percentage of clones for which the correct insertion was detected was even higher than for strain MG1655 Besides a higher background of unspecific insertion events for MG1655 we also observed that the two expression plasmids pRedET and 706 FLP persisted much longer at 37 C in MG1655 cells in comparison to W3110 For MG1655 and derived st
33. lanked by FRT sites and 50 bp long homology arms for the control experiment 400 ng ul 10 ul E coli cells pRedET tet Glycerol stock of E coli strain HS996 harboring the expression plasmid pRedET tet for the control experiment 500 ul 25 glycerol E coli cells q manXYZ kan Glycerol stock of the modified E coli strain HS996 The mannose transporter manXYZ is replaced by the FRT flanked kanamycin resistance marker cassette control experiment 500 yl 2596 glycerol PCR primer 1 Amplification primer located upstream of the manXYZ deletion to confirm the correct insertion of the FRT PGK gb2 neo FRT cassette in the E coli chromosome 10 uM 20 ul PCR primer 2 Amplification primer located on the FRT PGK gb2 neo FRT cassette to confirm its correct insertion in the E coli chromosome 10 uM 20 ul PCR primer 3 Amplification primer located on the FRT PGK gb2 neo FRT cassette to confirm its correct insertion in the E coli chromosome 10 uM 20 ul 10 PCR primer 4 Amplification primer located downstream of the manXYZ deletion to confirm the correct insertion of the FRT PGK gb2 neo FRT cassette in the E coli chromosome 10 uM 20 pl 11 This manual with protocols maps and sequences Please store tubes 1 4 and 7 10 at 20 C store tubes 5 and 6 at 80 C Please note All materials necessary for the control experiment are provided with this kit You must order your oligonucleotides PCR primers according to your
34. lin otherwise the Red ET Recombination protein expression plasmid pPRedET will either persist or the cells will die Incubate the plates at 37 C overnight The Red ET Recombination protein expression plasmid pRedET will disappear at 37 C You should obtain gt 100 colonies and the ration of induced uninduced bacterial colonies should exceed 10 1 Note Since a thin layer of unmodified E coli cells is generally present all over the plate and also beneath the obtained colonies the clones should be purified from these traces by streaking them out on a fresh LB agar plate containing kanamycin 15 pg ml Single colonies from this plate should then be analyzed by PCR to confirm the correct insertion of the cassette into the E coli chromosome Figure 4 Eight colonies were streaked out on a fresh plate to obtain single colonies which were then analyzed by PCR 15 Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 6 4 Verification of successfully modified genome by PCR analysis Analyze the obtained colonies by colony PCR e g pick a single colony and resuspend it in 30 ul of sterile water Boil the sample at 98 C for 5 minutes and take an aliquot of 2 ul of the suspension as template for your PCR reaction For the control experiment two pairs of primers are included tube 7 to 10 amplifying a 503bp fragment for the left border tube 7 and 8 and a 425bp fragment for the right border of the inserted
35. n or data use of the product for diagnostic purposes or re sale of the product or its components for any purpose commercial or otherwise The use of homologous recombination for commercial purposes may infringe the intellectual property covered by the EP 419 621 patent family Products containing the araB promoter are sold under patent license for research purposes only and are non transferable Inguiries for any commercial use including production of material to be sold commercially or used in production or in product development efforts which includes efforts toward regulatory approval should be made directly to Xoma Corporation Berkeley California Xoma Corporation 2910 Seventh Street Berkeley CA 94710 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 Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 30 10 Other products available from Gene Bridges General information q 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 the all information how to order the kits in your countr
36. nces pBAD gam beta pRedET 9270 bp pSC101 ori Figure 8 Map of the Red ET expression plasmid pRedET tet Transformation of E coli hosts with this plasmid is selected for by acquisition of tetracycline resistance at 30 C Expression of the Red ET Recombination proteins is induced by L arabinose activation of the BAD promoter at 37 C The second version of the expression plasmid supplied with the kit pRedET amp is identical but carries an ampicillin resistance marker instead of the tetracycline resistance gene CI 578 FLP 706 Flp tetR 11540 bp pSC101 ori 22 Figure 9 Map of the plasmid 706 FLP Transformation of E coli hosts with this plasmid is selected for by acquisition of tetracycline resistance at 30 C Expression of the FLPe recombination proteins is induced by a temperature shift to 37 C Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 20 Notl Xhol pGB2 kanamycin neomycin Not 1 AATTAACCCTCACTAAAGG GCGGCCGC GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC ATTCTACOGG 72 GTAGGGGAGG CGCTTTTCCC AAGGCAGTCT GGAGCATGCG CTTTAGCAGC CCCGCTGGGC ACTTGGCGCT 142 ACACAAGTGG CCTCTGGCCT CGCACACATT CCACATCCAC CGGTAGGCGC CAACCGGCTC CGTTCTTTGG 212 TGGCCCCTTC GCGCCACCTT CCACTCCTCC CCTAGTCAGG AAGTTCCCCC OCGOOOOGCA GCTCGCGTCG 282 TGCAGGACGT GACAAATGGA AGTAGCACGT CTCACTAGTC TCGTGCAGAT GGACAGCACC GCTGAGCAAT 352 GGAAGCGGGT AGGCCTTTGG GGCAGCGGCC AATAGCA
37. ns Introduction of site specific targeting sites loxP FRT etc Insertion of restriction enzyme recognition sites Subcloning of DNA pieces up to 60 kb Transferring DNA fragments into multiple destination vectors BAC and cosmid stitching Substitutions A A A A A A A A A Contact our DNA Engineering Service by email to contact genebridges com or go to www genebridges com for details and prices 37 Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 38 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
38. ntose phosphate pathway as well as the common aromatic amino acid pathway have to be manipulated The complexity of the necessary modifications reguires a tool allowing the precise knock out or alteration of multiple genes without leaving antibiotic selection markers Red ET recombination first published under the name ET cloning Zhang et al 1998 and also known as recombineering is an easy to use modification system for prokaryotic functional genomics Red ET Recombination relies on homologous recombination in vivo in E coli and allows a wide range of modifications of 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 These qualities are optimal for engineering a DNA molecule regardless of its size Homologous recombination occurs through homology regions which are stretches 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 specifically be 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 Zhang and coworkers demonstrated in 1998 for the first time that a pair of phage coded proteins RecE and RecT only need 42bp long homology arms to mediate the homologous recombination
39. or 30 sec at 11 000 rpm in a cooled microfuge benchtop centrifuge at 2 C Discard the supernatant by quickly tipping out the supernatant twice and place the pellet on ice Resuspend the pellet with 1 ml chilled ddH2O 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 Resuspend the cells and keep the tube on ice 5 Add 1 ul of the expression plasmid e g 706 FLP to your cell suspension Mix briefly and keep the tube on ice Transfer the cell suspension from the tube to the chilled electroporation cuvette 6 Electroporate at 1350 V 10 nf 600 Ohms This setting applies to an Eppendorf Electroporator 2510 using a 1 mm electroporation cuvette Other devices can be used but 1350 V and a 5 ms pulse are recommended 7 Resuspend the electroporated cells in 1 ml LB medium without antibiotics and return them to the microfuge tube 8 Incubate at 30 C for 70 min shaking at 1000 rpm The expression plasmid 706 FLP will be lost at 37 C 9 Using a small loop plate 100 ul cells on LB agar plates containing tetracycline 3 ug ml plus kanamycin 15 ug ml Incubate the plates at 30 C overnight or for at least 15 h Protect the plate from light by wrapping it up as tetracycline is sensitive to light Make sure the cells stay at 30 C otherwise the expression plasmid will
40. pression of genes mediating Red ET is induced by the addition of L arabinose After induction the cells are prepared for electroporation and the PCR product carrying the homology arms is electroporated step 3 Red ET recombination inserts the functional cassette into the target locus step 4 Only colonies carrying the inserted modification replacement of a gene by the FRT PGK gb2 neo FRT cassette will survive kanamycin selection on the agar plates Optionally the kanamycin selection marker can be removed from the chromosome by transforming the cells with FLP expression plasmid e g 706 FLP Gene Bridges Cat No A103 step 5 Expression of the site specific FLP recombinase removes the selection marker from the target locus leaving a single FRT site as footprint behind step 6 Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 6 3 How Red ET Recombination works In Red ET Recombination also referred to as mediated recombination target DNA molecules are precisely altered by homologous recombination in E coli which express the phage derived protein pairs either RecE RecT from the Rac prophage or Red Redb from phage These protein pairs are functionally and operationally equivalent RecE and RedU are 5 3 exonucleases and RecT and Redb are DNA annealing proteins A functional interaction between RecE and RecT or between RedU and Redb is also reguired in order to catalyze the homologous recom
41. quences is not needed this part can simply be omitted from the oligonucleotide design 3 Required Part C C fpr the second oligonucleotide This piece usually 18 to 24 nucleotides long primes the PCR amplification of the selectable marker from the provided template PCR template with annealed oligos PCR product amp Vector Targeting construct N B k C x 3 A A Sm Sm m Sm 3 C Be A B AN S N A N a N Vector Figure 3 Practical steps involved in Red ET 9 Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 The two oligos below were used to add the 50 bp homology regions for Red ET Recombination to the FRT PGK gb2 neo FRT cassette used in the control reaction Figure 11 The fragments of the oligos which serve as homology arms are shown in plain text Figure 3 Part A respectively A the parts which serve as PCR primers for amplification of the cassette are underlined Figure 3 Part C respectively C These two oligos are not supplied with the kit Oligo 1 upper oligo 5 GTTGATACATGGGGAGGCAGCCCGTTCAATGCTGCCAGCCGCATTGTCGT AATTAACCCTCACTAAAGGGCGG 3 Oligo 2 lower oligo 5 CGAGCATTGGAATGTTAACGCCTGCAATGACTTCATAATGCTCTTTGTCGTAA TACGACTCACTATAGGGCTCG 3 Oligonucleotide Design for your target gene I Choose 50 nucleotides N so directly adjacent upstream 5 to the intended insertion site Order an oligonucleotide with this sequence at the
42. rains a marker test LB LB tet similar to the one described for 706 FLP page 20 is highly recommended to confirm the loss of the pRedET plasmid Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 26 8 References and Patents 8 1 References Angrand P O Daigle N van der Hoeven F Scholer H R Stewart A F Simplified generation of targeting constructs using ET recombination Nucleic Acids Res 27 e16 1999 Datsenko K A and Wanner B L One step inactivation of chromosomal genes in Escherichia coli K 12 using PCR products PNAS 97 6640 6645 2000 Derbise A Lesic B Dacheux D Ghigo J M and Carniel E A rapid and simple method for inactivating chromosomal genes in Yersinia FEMS 38 113 116 2003 1 Guzman L M Belin D Carson M J Beckwith J Tight regulation modulation and high level expression by vectors containing the arabinose pBAD promoter J Bacteriol 177 4121 4130 1995 Kilby N J Snaith M R and Murray J A Site specific recombinases tools for genome engineering Trends Genet 9 413 421 1993 Miller C A Ingmer H and Cohen SN Boundaries of the pSC101 Minimal Replicon are Conditional J Bacteriol 177 4865 4871 1995 Mundy R Petrovska L Smollett K Simpson N Wilson R K Yu J Tu X Rosenshine I Clare S Dougan G and Frankel G Identification of a novel Citrobacter rodentium type Ill secreted protein espl and roles of this an
43. ro Ala Thr Oys Pro Phe Asp His Gin Ala Lys His Arg Ile Glu Arg 1090 GCA OGT ACT CGG ATG GAA GOC GGT CTT GTC GAT CAG GAT GAT CTG GAC GAA GAG CAT 147 Ala Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gin Asp Asp Leu Asp Glu Glu His 1147 CAG 33 CTC COG CCA GCC GAA CTG TTC GOC AGG CTC AAG GOS CGC ATG COC GAC GGC 166 Gin Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala Arg Met Pro Asp Gly 1204 GAG GAT CTC GTC GTG ACC CAT GOC GAT GOC TGC TTG COG AAT ATC ATG GTG GAA AAT 185PGlu Asp Leu Val Val Thr His Gly Asp Ala Os Leu Pro Asn Ile Met Val Glu Asn 1261 GGC CSC TIT TCT GGA TTC ATC GAC TOT GGC OGG CTG GGT GTG GOG GAC OGC TAT CAS 204b Gly Arg Phe Ser Gly Phe Ile Asp Os Gly Arg Leu Gly Val Ala Asp Arg Tyr Gin 1318 GAC ATA GOG TTG GCT ACC CGT GAT ATT GOT GAA GAG CIT GIC GGC GAA TOG GCT GAC 223b Asp Ile Ala Leu Ala Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp 1375 CGC TTC CIC GIG CIT TAC GGT ATC GOC SCT CCU GAT TOG CAG OGC ATC GCC TTC TAT 242P Arg Phe Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gin Arg Ile Ala Phe Tyr 1432 OS CTT CTT GAC GAG TTC TTC TGA GCGGGACTCTGGGGTTCGAATAAAGACCGACCAAGCGAC GTC 261b Arg Leu Leu Asp Glu Phe Phe 1498 TGA GAGCTCCCTG GOGAATTOGG TACCAATAAA AGAGCTTTAT TTTCATGATC TGTGTGTTGG Xhol 1561 TTTTTGTGTG CGGCGCG GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC C TCGAGCCCTATAGTGAGTCGT 1634 ATTA Figure 10 Map of the FRT PGK gb2 neo FRT cassette 21 Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 1 GIGACCATTGCT
44. tant by quickly tipping out the supernatant twice and place the pellet on ice Resuspend the pellet with 1 ml chilled ddH2O 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 Resuspend cells and keep the tube on ice 4 Take the Red ET Recombination protein expression plasmid pRedET tube 1 Add 1 ul to your cell pellet Mix briefly Keep the tube on ice Transfer the cell suspension from the tube to the chilled electroporation cuvette 5 Electroporate at 1350 V 10nfr 600 Ohms This setting applies to an Eppendorf Electroporator 2510 using a 1 mm electroporation cuvette Other devices can be used but 1350 V and a 5 ms pulse are recommended 6 Resuspend the electroporated cells in 1 ml LB medium without antibiotics and return them to the microfuge tube 7 Incubate at 30 C for 70 min shaking at 1000 rpm The Red ET expression plasmid pRedET will be lost at 37 C 8 Using a small loop plate 100 ul cells on LB agar plates containing tetracycline 3 ug ml or ampicillin 50 ug ml depending on which pRedET plasmid you used in step 4 Use a loop to streak the control culture tube 5 E coli cells pRedET on an LB agar plate with tetracycline 3 ng ml Incubate the plates at 13 Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 30
45. trifuge 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 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 Resuspend cells and keep the tubes on ice 5 Add 1 2 ul 400 800 ng of your prepared linear FRT PGK gb2 neo FRT fragment with homology arms to the pellet to each of the two microfuge tubes induced and uninduced and pipette the mixture into the chilled electroporation cuvettes In parallel pipette 2 ul from tube 4 into each of the two tubes of the control 6 Electroporate at 1350 V 10 nf 600 Ohms This setting applies to an Eppendorf Electroporator 2510 using an electroporation cuvette with a slit of 1mm Other devices can be used but 1350 V and a 5 ms pulse are recommended 7 Add 1 LB medium 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 3 hours Recombination will now occur 8 Spin down the whole culture for 30 seconds and remove 900yl of the supernatant Resuspend the cells in the remaining medium and streak out the cultures with a loop onto LB agar plates containing kanamycin 15 ug ml The plates should not contain tetracycline or ampicil
46. uge tubes 2 for your own experiment and 2 for control experiment containing 1 4 ml fresh LB medium conditioned with the same antibiotics as in step 1 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 Incubate the tubes at 30 C for approximately 2 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 arabinose Leave the other tubes without induction as negative controls Incubate all at 37 C shaking for 45 min to 1 h Note It is important that cells are incubated at 37 C the temperature at which all proteins necessary for the subsequent recombination are expressed There are about 5 copies of this temperature sensitive plasmid per cell and during one hour there is approximately 1 doubling step meaning any daughter cell will still have on average 2 3 copies left and will also go on expressing the recombination proteins The plasmid is actually lost after electroporation and recombination when cells are incubated at 37 C overnight Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 14 4 Prepare the cells for electroporation Centrifuge for 30 sec at 11 000 rpm in a cooled microfuge benchtop cen
47. y is given on our website www genebridges com K001 Auick and Easy BAC Modification Kit Description Contents A A This kit is designed to modify any type of bacterial artificial chromosomes BACs within 1 2 weeks by using a Kanamycin Neomycin cassette This kit is optimized for basic modifications such as insertions or deletions of fragments in any type of bacterial artificial chromosomes BACs leaving a selectable marker gene This kit can also be used to work on bacterial chromosomes and common ColE1 origin plasmids High Red ET efficiency plus convenient removal of the Red ET Recombination protein expression plasmid pRedET after recombination Red ET Recombination protein expression plasmid pRedET Any E coli strain can be made Red ET proficient by transformation with this plasmid BAC host E coli strain HS996 already carrying the Red ET plasmid Tn5 neomycin resistance template to be used for your own experiments Positive controls to introduce a Tn5 neo cassette in a 150 kb BAC Detailed protocols descriptions of plasmids maps and seguences 31 Gene Bridges Quick and Easy E coli Gene Deletion Kit Version 2 0 January 2007 K002 Counter Selection BAC Modification Kit Description Contents 1 A 9 9 A This kit is designed to modify any type of bacterial artificial chromosomes BACs within 2 3 weeks by using a counter selection cassette The kit is designed for advanced BAC modification such
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