recombineering



Recombineering is a highly efficient and precise molecular cloning method used often for the engineering of large or difficult to engineer sequences such as the generation of seamlessly mutated bacterial artificial chromosomes (BACs) including insertion of point mutations, protein tags, landing pads and reporter genes.  NovoHelix's recombineering service offers counterselection approaches for introduction of point mutants, inactivation of non-relevant genes on the BAC to prevent overexpression, gene replacement for humanization and BAC reduction by ‘shaving’ tens of kilobases. Our recombineering portfolio consists of successful designs of complex genetic circuits, incorporation of site-specific recombinases such as Cre/CreERt2, flippase (Flp/Flpe/Flpo), Dre, VCre, Vika, Nigri & Panto, usage of heterotypic recombinase sites such as lox66/lox2272, Frt3/Frt5/Frt14/15, rox/rox12, recycling of antibiotic cassettes for selection with ampicillin (Amp), blasticidin (Bsd), carbenicillin (Carb), chloramphenicol (Cm), gentamycin (Gent), hygromycin (Hygro), kanamycin (Kan), nourseothricin (clonNAT), puromycin (Puro), spectinomycin (Spec), streptomycin (Strep), tetracycline (Tet), trimethoprim (Tmp) and zeocin (Zeo)/bleocin (Bleo)/phleomycin (Phleo). In short, NovoHelix is an industry leader and has performed many challenging recombineering experiments. Please contact us for a custom quote or see our Animal Models – BAC recombineering pages for mouse or rat BAC reporter animals.
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Service

Catalog Nr

Service Description

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BAC recombineering, basic (up to 2 steps)
MBRC001
Recombineering is a highly efficient and precise method used for the generation of seamlessly mutated bacterial artificial chromosomes (BACs) including insertion of point mutations, protein tags, landing pads and reporter genes. NovoHelix's BAC recombineering service offers counterselection approaches, inactivation of non-relevant genes on the BAC to prevent overexpression, gene replacement for humanization and BAC reduction by shaving.  Our recombineering portiolo consists of successful design of complex genetic circuits, incorporation of site-specific recombinases such as Cre, CreERt2, flippase (Flp/Flpe/Flpo), Dre, VCre, Vika, Nigri & Panto, usage of heterotypic recombinase sites such as lox66/lox2272, Frt3/Frt5/Frt14/15, rox/rox12, recycling of antibiotic cassettes for selection with ampicillin (Amp), blasticidin (Bsd), carbenicillin (Carb), chloramphenicol (Cm), gentamycin (Gent), hygromycin (Hygro), kanamycin (Kan), nourseothricin (clonNAT), puromycin (Puro), spectinomycin (Spec), streptomycin (Strep), tetracycline (Tet), trimethoprim (Tmp) and zeocin (Zeo)/bleocin (Bleo)/phleomycin (Phleo). We include up to 2 recombineering steps for our basic service.  Basic service includes selection with ampicillin, carbenicillin, chloramphenicol, kanamycin, spectinomycin, tetracycline or trimethoprim. Additional charges may incur for more recombineering steps and selection with antibiotics not included in our basic service.  The modified BAC will be provided in the E. coli strain as a glycerol stock or stab culture.


BAC recombineering - advanced
MBRC002
Advanced recombineering service includes steps that supercede 3 or more recombineering reactions and includes selection steps with non-standard antibiotics such as blasticidin, gentamycin, hygromycin, nourseothricin (clonNAT), puromycin, streptomycin and zeocin/bleocin/phleomycin or others. The advanced recombineering service route also includes seamless knockin of large cassettes > 3 kb and development of screening assays for E. coli specific-strain generation.  NovoHelix has pioneered BAC shaving and deleted tens of kilobases from an existing BAC to remove extraneous sequences and genes linked on the same chromosome. The modified BAC will be provided in the E. coli strain as a glycerol stock or stab culture. 
BAC purification & validation
MBRC003
Purification of supercoiled BACs by a proprietary anion exchange (AEX) chromatography, validation by PCR screening around the BAC at ~20-kb intervals and validation by restriction digestion and analysis by pulse-field gel electrophoresis (PFGE). Endotoxin levels are generally very low (0.05  – 0.5 ng LPS/µg) via our proprietary extraction method and are adequate for sensitive applications such as zygotic microinjection of mammalian embryos. The BAC is predominantly in its supercoiled topology and free of RNA and protein contamination.  Linearization with a homing nuclease such as PI-SceI and drop dialysis in 1x TE or microinjection buffer is included in the purification service. Validation by PCR screening at higher resolution (5-kb, 10-kb) or additional restriction digestions (greater than 3) is available with commensurate fee schedule.
technology
references
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Zhang Y, Buchholz F, Muyrers JP, Stewart AF. A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet. 1998 Oct;20(2):123-8. PubMed PMID: 9771703.


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Poser I, Sarov M, Hutchins JR, Hériché JK, Toyoda Y, Pozniakovsky A, Weigl D,  Nitzsche A, Hegemann B, Bird AW, Pelletier L, Kittler R, Hua S, Naumann R, Augsburg M, Sykora MM, Hofemeister H, Zhang Y, Nasmyth K, White KP, Dietzel S, Mechtler K, Durbin R, Stewart AF, Peters JM, Buchholz F, Hyman AA. BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals. Nat Methods. 2008 May;5(5):409-15. doi: 10.1038/nmeth.1199. Epub 2008 Apr 6. Erratum in: Nat Methods. 2008 Aug;5(8):748. PubMed PMID: 18391959; PubMed Central PMCID: PMC2871289.


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Rogatcheva MM, Rund LA, Swanson KS, Marron BM, Beever JE, Counter CM, Schook LB. Creating porcine biomedical models through recombineering. Comp Funct Genomics. 2004;5(3):262-7. doi: 10.1002/cfg.404. PubMed PMID: 18629152; PubMed Central PMCID: PMC2447442.


Warming S, Costantino N, Court DL, Jenkins NA, Copeland NG. Simple and highly  efficient BAC recombineering using galK selection. Nucleic Acids Res. 2005 Feb 24;33(4):e36. PubMed PMID: 15731329; PubMed Central PMCID: PMC549575.


Bird AW, Erler A, Fu J, Hériché JK, Maresca M, Zhang Y, Hyman AA, Stewart AF.  High-efficiency counterselection recombineering for site-directed mutagenesis in  bacterial artificial chromosomes. Nat Methods. 2011 Dec 4;9(1):103-9. doi: 10.1038/nmeth.1803. PubMed PMID: 22138824.


Chan W, Costantino N, Li R, Lee SC, Su Q, Melvin D, Court DL, Liu P. A recombineering based approach for high-throughput conditional knockout targeting  vector construction. Nucleic Acids Res. 2007;35(8):e64. Epub 2007 Apr 10. PubMed  PMID: 17426124; PubMed Central PMCID: PMC1885671.


Fu J, Bian X, Hu S, Wang H, Huang F, Seibert PM, Plaza A, Xia L, Müller R, Stewart AF, Zhang Y. Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting. Nat Biotechnol.  2012 May;30(5):440-6. doi: 10.1038/nbt.2183. PubMed PMID: 22544021.


Thomason LC, Costantino N, Court DL. Examining a DNA Replication Requirement for Bacteriophage λ Red- and Rac Prophage RecET-Promoted Recombination in Escherichia coli. MBio. 2016 Sep 13;7(5). pii: e01443-16. doi:10.1128/mBio.01443-16. PubMed PMID: 27624131; PubMed Central PMCID: PMC5021808.


Egbert RG, Rishi HS, Adler BA, McCormick DM, Toro E, Gill RT, Arkin AP. A versatile platform strain for high-fidelity multiplex genome editing. Nucleic Acids Res. 2019 Apr 8;47(6):3244-3256. doi: 10.1093/nar/gkz085. PubMed PMID: 30788501; PubMed Central PMCID: PMC6451135.


Pines G, Freed EF, Winkler JD, Gill RT. Bacterial Recombineering: Genome Engineering via Phage-Based Homologous Recombination. ACS Synth Biol. 2015 Nov 20;4(11):1176-85. doi: 10.1021/acssynbio.5b00009. Epub 2015 Apr 27. Review. PubMed PMID: 25856528.


Reynolds TS, Gill RT. Quantifying Impact of Chromosome Copy Number on Recombination in Escherichia coli. ACS Synth Biol. 2015 Jul 17;4(7):776-80. doi:  10.1021/sb500338g. Epub 2015 Mar 19. PubMed PMID: 25763604.


Wannier TM, Nyerges A, Kuchwara HM, Czikkely M, Balogh D, Filsinger GT, Borders NC, Gregg Cj, Lajoie MJ, Rios X, Pal C, Church GM. Improved bacterial recombineering by parallelized protein discovery. bioRxiv. 2020 Jan 16.  doi: 10.1101/2020.01.14.906594.

Chuang K, Nguyen E, Sergeev Y, Badea TC. Novel Heterotypic Rox Sites for Combinatorial Dre Recombination Strategies. G3 (Bethesda). 2015 Dec 29;6(3):559-71. doi: 10.1534/g3.115.025841. PubMed PMID: 26715092; PubMed Central PMCID: PMC4777119.

Rondelet A, Pozniakovsky A, Leuschner M, Poser I, Ssykor A, Berlitz J, Schmidt N,  Hyman AA, Bird AW. ESI mutagenesis: A one-step method for introducing point  mutations into bacterial artificial chromosome transgenes. bioRiv. doi: 10.1101/844282 Epub 2020 Apr 23. https://www.biorxiv.org/content/10.1101/844282v2

Gregg CJ, Lajoie MJ, Napolitano MG, Mosberg JA, Goodman DB, Aach J, Isaacs FJ, Church GM. Rational optimization of tolC as a powerful dual selectable marker for genome engineering. Nucleic Acids Res. 2014 Apr;42(7):4779-90. doi: 10.1093/nar/gkt1374. Epub 2014 Jan 22. PubMed PMID: 24452804; PubMed Central PMCID: PMC3985617.

Li XT, Thomason LC, Sawitzke JA, Costantino N, Court DL. Positive and negative selection using the tetA-sacB cassette: recombineering and P1 transduction in Escherichia coli. Nucleic Acids Res. 2013 Dec;41(22):e204. doi: 10.1093/nar/gkt1075. Epub 2013 Nov 6. PubMed PMID: 24203710; PubMed Central PMCID: PMC3905872.

Wang H, Bian X, Xia L, Ding X, Müller R, Zhang Y, Fu J, Stewart AF. Improved seamless mutagenesis by recombineering using ccdB for counterselection. Nucleic Acids Res. 2014 Mar;42(5):e37. doi: 10.1093/nar/gkt1339. Epub 2013 Dec 24. PubMed PMID: 24369425; PubMed Central PMCID: PMC3950717.
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