Abstract
Strain engineering, like cloning, is a fundamental technology used to confer new traits onto existing strains. While effective methods for trait development through gene modification within strains have been developed, methods for trait transfer between Escherichia coli strains to create complex strains are needed. We report herein the development of genome mass transfer (GMT), a broadly applicable new strain engineering methodology enabling rapid trait transfer from a donor strain into a recombineering gene-expressing recipient strain. GMT utilizes electroporation of donor chromosomal DNA into a recombineering recipient cell for precise trait transfer. GMT transfer of traits between E. coli strains can be used to rapidly assemble new strains incorporating combinations of marked gene knockouts, for example, utilizing the existing E. coli K-12 Keio gene knockout collection as source target genes. Optional use of random primed isothermal amplified DNA eliminates the need for initial DNA purification, affording high throughput application. This allows unprecedented simplicity and speed for rational design engineering of complex phenotypes in industrial strains.
Similar content being viewed by others
References
Alper, H., Miyaoku, K., & Stephanopoulos, G. (2005). Construction of lycopene-overproducing E. coli strains by combining systematic and combinatorial gene knockout targets. Nature Biotechnology, 5, 612–616.
Thomason, L. C., Costantino, M., & Court, D. L. (2007). E. coli genome manipulation by P1 transduction. Current protocols in molecular biology (Chapter 1, Unit 1.17). NY: Wiley.
Sternberg, N., & Hoess, R. (1983). The molecular genetics of bacteriophage P1. Annual Review of Genetics, 17, 123–154.
Goodson, M., & Rowbury, R. J. (1987). Altered phage P1 attachment to strains of Escherichia coli carrying the plasmid ColV, I-K94. Journal of General Virology, 68, 1785–1789.
McKane, M., & Milkman, R. (1995). Transduction, restriction and recombination patterns in Escherichia coli. Genetics, 139, 35–43.
Bachmann, B. J. (1990). Linkage map of Escherichia coli K-12, Edition 8. Microbiological Reviews, 54, 130–197.
Murphy, K. C. (1998). Use of bacteriophage λ recombination functions to promote gene replacement in Escherichia coli. Journal of Bacteriology, 180, 2063–2071.
Datsenko, K. A., & Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences of the United States of America, 97, 6640–6645.
Yu, D., Ellis, H. M., Lee, E. C., Jenkins, N. A., Copeland, N. G., & Court, D. L. (2000). An efficient recombination system for chromosome engineering in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, 97, 5978–5983.
Zhang, Y., Buchholz, F., Muyrers, J. P., & Stewart, A. F. (1998). A new logic for DNA engineering using recombination in Escherichia coli. Nature Genetics, 20, 123–128.
Court, D. L., Sawitzke, J. A., & Thomason, L. C. (2002). Genetic engineering using homologous recombination. Annual Review of Genetics, 36, 361–388.
Sarov, M., Schneider, S., Pozniakovski, A., Roquev, A., Ernst, S., Zhang, Y., et al. (2006). A recombineering pipeline for functional genomics applied to Caenorhabditis elegans. Nature Methods, 3, 839–844.
Datta, S., Costantino, N., & Court, D. L. (2006). A set of recombineering plasmids for gram-negative bacteria. Gene, 379, 109–115.
Wang, J., Sarov, M., Rientjes, J., Fu, J., Hollak, H., Kranz, H., et al. (2006). An improved recombineering approach by adding RecA to lambda Red recombination. Molecular Biotechnology, 32, 43–53.
Baba, T., Ara, T., Haseqawa, M., Takai, Y., Okumura, Y., Baba, M., et al. (2006). Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Molecular Systems Biology, 2, 2006.0008.
Gerdes, S. Y., Scholle, M. D., Campbell, J. W., Balazsi, G., Ravasz, E., Daugherty, M. D., et al. (2003). Experimental determination and system level analysis of essential genes in Escherichia coli MG1655. Journal of Bacteriology, 185, 5673–5684.
Kang, Y., Durfee, T., Glasner, J. D., Qiu, Y., Frish, D., Winterberg, K. M., et al. (2004). Systematic mutagenesis of the Escherichia coli genome. Journal of Bacteriology, 186, 4921–4930.
Patnaik, R. (2008). Engineering complex phenotypes in industrial strains. Biotechnology Progress, 24, 38–47.
Carnes, A. E., Hodgson, C. P., & Williams, J. A. (2007). Improved E. coli plasmid production strains. US Patent Application 60931465.
Williams, J. A. (2008). Processes for improved strain engineering. World Patent Application WO2008153731.
Williams, J. A. (2008). Vectors and methods for genetic immunization. World Patent Application WO2008153733.
Haldimann, A., & Wanner, B. L. (2001). Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-function studies of bacteria. Journal of Bacteriology, 183, 6384–6393.
Imam, A. M., Patrinos, G. P., de Krom, M., Bottardi, S., Janssens, R. J., Katsantoni, E., et al. (2000). Modification of human β-globin locus PAC clones by homologous recombination in Escherichia coli. Nucleic Acids Research, 28, e65.
Ausubel, F. M. (1998). Current protocols in molecular biology. NY: Wiley.
Jia, X., Kostal, J., & Claypool, J. A. (2006). Controlled lysis of bacteria. US Patent Application 2006/0040393.
Choi, K. H., Kumar, A., & Schweizer, H. P. (2005). A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: Application for DNA fragment transfer between chromosomes and plasmid transformation. Journal of Microbiol Methods, 64, 391–397.
Charles, T. C., Doty, S. L., & Nester, E. W. (1994). Construction of Agrobacterium strains by electroporation of genomic DNA and its utility in analysis of chromosomal virulence mutations. Applied and Environmental Microbiology, 60, 4192–4194.
Cohen, A., & Clark, A. J. (1986). Synthesis of linear plasmid multimers in Escherichia coli K-12. Journal of Bacteriology, 167, 327–335.
De Vito, J. A. (2008). Recombineering with tolC as a selectable/counter-selectable marker: remodeling the rRNA operons of Escherichia coli. Nucleic Acids Research, 36, e4.
Wong, Q. N., Ng, V. C., Lin, M. C., Kung, H. F., Chan, D., & Huang, J. D. (2005). Efficient and seamless DNA recombineering using a thymidylate synthase A selection system in Escherichia coli. Nucleic Acids Research, 33, e59.
Warming, S., Costantino, N., Court, D. L., Jenkins, N. A., & Copeland, N. G. (2005). Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Research, 33, e36.
Wang, S., Zhao, Y., Leiby, M., & Zhu, J. (2009). A new positive/negative selection scheme for precise BAC recombineering. Molecular Biotechnology, 42, 110–116.
Murphy, K. C., & Campellone, K. G. (2003). Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli. BMC Molecular Biology, 4, 11.
Poteete, A. R. (2001). What makes the bacteriophage λ Red system useful for genetic engineering: Molecular mechanism and biological function. FEMS Microbiology Letters, 201, 9–14.
Baitin, D. M., Bakhlanova, I. V., Kil, Y. V., Cox, M. M., & Lanzov, V. A. (2006). Distinguishing characteristics of hyperrecombinogenic RecA protein from Pseudomonas aeruginosa acting in Escherichia coli. Journal of Bacteriology, 188, 5812–5820.
Lambertsen, L., Sternberg, C., & Molin, S. (2004). Mini-Tn7 transposons for site-specific tagging of bacteria with fluorescent proteins. Environmental Microbiology, 6, 726–732.
Cosloy, D. S., & Oishi, M. (1973). Genetic transformation in Escherichia coli K12. Proceedings of the National Academy of Sciences of the United States of America, 70, 84–87.
Yu, B. J., Sung, B. H., Koob, M. D., Lee, C. H., Lee, J. H., Lee, W. S., et al. (2002). Minimalization of the Escherichia coli genome using a Tn5-targeted Cre/loxP excision system. Nature Biotechnology, 20, 1018–1023.
Alper, H., & Stephanopoulos, G. (2008). Uncovering the gene knockout landscape for improved lycopene production in E. coli. Applied Microbiology and Biotechnology, 78, 801–810.
Meynial-Salles, I., Cervin, M. A., & Soucaille, P. (2005). New tool for metabolic pathway engineering in Escherichia coli: One-step method to modulate expression of chromosomal genes. Applied and Environmental Microbiology, 71, 2140–2144.
Braatsch, S., Helmark, S., Kranz, H., Koebmann, B., & Jensen, P. R. (2008). Escherichia coli strains with promoter libraries constructed by Red/ET recombination pave the way for transcriptional fine-tuning. Biotechniques, 45, 335–337.
Posfai, G., Plunkett, G., I. I. I., Feher, T., Frisch, D., Keil, G. M., Umenhoffer, K., et al. (2006). Emergent properties of reduced-genome Escherichia coli. Science, 312, 1044–1046.
Fu, J., Wenzel, S. C., Perlova, O., Wang, J., Gross, F., Tang, Z., et al. (2008). Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition. Nucleic Acids Research, 36, e113.
Costantino, N., & Court, D. L. (2003). Enhanced levels of λ Red-mediated recombinants in mismatch repair mutants. Proceedings of the National Academy of Sciences of the United States of America, 100, 15748–15753.
Van Kessel, J. C., & Hatfull, G. F. (2007). Recombineering in Mycobacterium tuberculosis. Nature Methods, 4, 147–152.
Datta, S., Costantino, N., Zhou, X., & Court, D. L. (2008). Identification and analysis of recombineering functions from Gram-negative and Gram-positive bacteria and their phages. Proceedings of the National Academy of Science, 105, 1626–1631.
Acknowledgment
We thank Kim Hansen for technical assistance with plasmid preparations and PCR screening, and Jean-Pierre Bouche for kindly providing strain JS1910 containing ydeA (miniTet, tetR).
Funding
This study was supported by the National Institute of Health (R44GM072141 to J.A.W.).
Conflict of interest statement
J.A.W., J.L., and C.P.H. have an equity interest in Nature Technology Corporation.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Williams, J.A., Luke, J. & Hodgson, C. Strain Engineering by Genome Mass Transfer: Efficient Chromosomal Trait Transfer Method Utilizing Donor Genomic DNA and Recipient Recombineering Hosts. Mol Biotechnol 43, 41–51 (2009). https://doi.org/10.1007/s12033-009-9177-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12033-009-9177-5