Strain Engineering by Genome Mass Transfer: Efficient Chromosomal Trait Transfer Method Utilizing Donor Genomic DNA and Recipient Recombineering Hosts

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.

Ecotin, a microbial inhibitor of serine proteases, blocks multiple complement dependent and independent microbicidal activities of human serum

“…The ecotin gene of E. coli BL21 (DE3) Star (Invitrogen), ATCC 12014 and ATCC 23505 (American Type Culture Collection, ATCC) was replaced with the chloramphenicol acetyltransferase gene (EC 2.3.1.28) using the recombineering plasmid pKD46-RecA as described [53,57]. The plasmid was electroporated into the bacteria, recombinant clones were isolated and the loss of the heat-sensitive plasmid was verified.…”

Bacterial Recombineering: Genome Engineering via Phage-Based Homologous Recombination

“…DSB may also be induced by CRISPR-Cas9 to cleave unmodified DNA following transformation with recombineering cassettes to eliminate wild-type background (29 , 30 ). If a selectable marker is included, a shotgun transformation of DNA can be used to rapidly modify a range of host traits (31 ). Although these approaches to recombineering rely on short homologies introduced by PCR or chemical synthesis, many bacteria will also integrate large fragments of DNA using their native recombination machinery (32 ).…”

“…29,30 If a selectable marker is included, then a shotgun transformation of DNA can be used to rapidly modify a range of host traits. 31 Although these approaches to recombineering rely on short homologies introduced by PCR or chemical synthesis, many bacteria will also integrate large fragments of DNA using their native recombination machinery. 32 This ability is exploited by conjugation-based approaches for genome engineering, where significant portions of the E. coli genome are transferred in a short time frame to rapidly generate diverse libraries using native recombination machinery.…”

Plasmid DNA fermentation strain and process‐specific effects on vector yield, quality, and transgene expression

“…Chromosome engineering to delete the ackA-pta and poxB genes was performed using lambda red recombination as described (Datsenko and Wanner, 2000) using the arabinose-inducible pKD46-recA or pKD46-recA PA recombineering plasmids (Williams et al, 2009b). A chloramphenicol resistance (Cm R ) marker was PCR-amplified from the R6K origin conditional replication plasmid pKD3 using 50 bp homology gene targeting primers.…”

“…The kan R marker was then excised. NTC48107 (DH5a dcm) and NTC48165 (DH5a dcm att l ::P 5/6 6/6 -RNA-IN-SacB, Cm R ) were made by electroporating dcm-735::kan containing Keio strain JW1944-2 (E. coli Genetic Stock Center) genomic DNA [genome mass transfer (GMT); Williams et al, 2009b] into DH5a or NTC4862 cells, respectively, containing pKD46-recA. The kan R marker was then excised.…”