Donald L. Court

Simple and highly efficient BAC recombineering using galK selection

Recombineering allows DNA cloned in Escherichia coli to be modified via lambda (λ) Red-mediated homologous recombination, obviating the need for restriction enzymes and DNA ligases to modify DNA. Here, we describe the construction of three new recombineering strains (SW102, SW105 and SW106) that allow bacterial artificial chromosomes (BACs) to be modified using galK positive/negative selection. This two-step selection procedure allows DNA to be modified without introducing an unwanted selectable marker at the modification site. All three strains contain an otherwise complete galactose operon, except for a precise deletion of the galK gene, and a defective temperature-sensitive λ prophage that makes recombineering possible. SW105 and SW106 cells in addition carry l-arabinose-inducible Cre or Flp genes, respectively. The galK function can be selected both for and against. This feature greatly reduces the background seen in other negative-selection schemes, and galK selection is considerably more efficient than other related selection methods published. We also show how galK selection can be used to rapidly introduce point mutations, deletions and loxP sites into BAC DNA and thus facilitate functional studies of SNP and/or disease-causing point mutations, the identification of long-range regulatory elements and the construction of conditional targeting vectors.

Recombineering: a powerful new tool for mouse functional genomics

Highly efficient phage-based Escherichia coli homologous recombination systems have recently been developed that enable genomic DNA in bacterial artificial chromosomes to be modified and subcloned, without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombinogenic engineering or recombineering, is efficient and greatly decreases the time it takes to create transgenic mouse models by traditional means. Recombineering also facilitates many kinds of genomic experiment that have otherwise been difficult to carry out, and should enhance functional genomic studies by providing better mouse models and a more refined genetic analysis of the mouse genome.

Recombineering: A Homologous Recombination-Based Method of Genetic Engineering

Recombineering is an efficient method of in vivo genetic engineering applicable to chromosomal as well as episomal replicons in Escherichia coli. This method circumvents the need for most standard in vitro cloning techniques. Recombineering allows construction of DNA molecules with precise junctions without constraints being imposed by restriction enzyme site location. Bacteriophage homologous recombination proteins catalyze these recombineering reactions using double- and single-stranded linear DNA substrates, so-called targeting constructs, introduced by electroporation. Gene knockouts, deletions and point mutations are readily made, gene tags can be inserted and regions of bacterial artificial chromosomes or the E. coli genome can be subcloned by gene retrieval using recombineering. Most of these constructs can be made within about 1 weeks time.

High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides.

Homologous DNA recombination is a fundamental, regenerative process within living organisms. However, in most organisms, homologous recombination is a rare event, requiring a complex set of reactions and extensive homology. We demonstrate in this paper that Beta protein of phage λ generates recombinants in chromosomal DNA by using synthetic single-stranded DNAs (ssDNA) as short as 30 bases long. This ssDNA recombination can be used to mutagenize or repair the chromosome with efficiencies that generate up to 6% recombinants among treated cells. Mechanistically, it appears that Beta protein, a Rad52-like protein, binds and anneals the ssDNA donor to a complementary single-strand near the DNA replication fork to generate the recombinant. This type of homologous recombination with ssDNA provides new avenues for studying and modifying genomes ranging from bacterial pathogens to eukaryotes. Beta protein and ssDNA may prove generally applicable for repairing DNA in many organisms.

Enhanced levels of λ Red-mediated recombinants in mismatch repair mutants

Homologous recombination can be used to generate recombinants on episomes or directly on the Escherichia coli chromosome with PCR products or synthetic single-stranded DNA (ssDNA) oligonucleotides (oligos). Such recombination is possible because bacteriophage λ-encoded functions, called Red, efficiently recombine linear DNA with homologies as short as 20–70 bases. This technology, termed recombineering, provides ways to modify genes and segments of the chromosome as well as to study homologous recombination mechanisms. The Red Beta function, which binds and anneals ssDNA to complementary ssDNA, is able to recombine 70-base oligos with the chromosome. In E. coli, methyl-directed mismatch repair (MMR) can affect these ssDNA recombination events by eliminating the recombinant allele and restoring the original sequence. In so doing, MMR can reduce the apparent recombination frequency by >100-fold. In the absence of MMR, Red-mediated oligo recombination can incorporate a single base change into the chromosome in an unprecedented 25% of cells surviving electroporation. Our results show that Beta is the only bacteriophage function required for this level of recombination and suggest that Beta directs the ssDNA to the replication fork as it passes the target sequence.

E. coli Genome Manipulation by P1 Transduction

This unit describes the procedure used to move portions of the E. coli genome from one genetic variant to another. Fragments of approximately 100 kb can be transferred by the P1 bacteriophage. The phage is first grown on a strain containing the elements to be moved, and the resulting phage lysate is used to infect a second recipient strain. The lysate will contain bacterial DNA as well as phage DNA, and genetic recombination, catalyzed by enzymes of the recipient strain, will incorporate the bacterial fragments into the recipient chromosome.

Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination

The bacterial chromosome and plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage‐encoded recombination functions efficiently to recombine sequences with homologies as short as 35 to 40 bases. This recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with dsDNA or ssDNA. Support protocols describe a two‐step method of making genetic alterations without leaving any unwanted changes, and a method for retrieving a genetic marker (cloning) from the E. coli chromosome or a co‐electroporated DNA fragment and moving it onto a plasmid. A method is also given to screen for unselected mutations. Additional protocols describe removal of defective prophage, methods for recombineering.

Transcription antitermination: the λ paradigm updated

Coliphage λ employs systems of transcription termination and antitermination to regulate gene expression. Early gene expression is regulated by the phage‐encoded N protein working with a series of Escherichia coli proteins, Nus, at RNA sites, NUT, to modify RNA polymerase to a termination‐resistant form. Expression of λ late genes is regulated by the phage‐encoded Q antitermination protein. Q, which appears to use only one host factor, acts at a DNA site, qut, to modify RNA polymerase to a termination‐resistant form. This review focuses on recent studies which show that: (i) N can mediate antitermination in vitro, independent of Nus proteins, (ii) Early genes in another lambdoid phage HK022 are also regulated by antitermination, where only an RNA signal appears necessary and sufficient to create a termination‐resistant RNA polymerase. (iii) A part of the qut signal appears to be read from the non‐template DNA strand. (iv) A host‐encoded inhibitor of N antitermination appears to act through the NUT site as well as with the α subunit of RNA polymerase, and is antagonized by NusB protein.

Rapid engineering of bacterial artificial chromosomes using oligonucleotides

Summary: A rapid method obviating the use of selectable markers to genetically manipulate large DNA inserts cloned into bacterial artificial chromosomes is described. Mutations such as single‐base changes, deletions, and insertions can be recombined into a BAC by using synthetic single‐stranded oligonucleotides as targeting vectors. The oligonucleotides include the mutated sequence flanked by short homology arms of 35–70 bases on either side that recombine with the BAC. In the absence of any selectable marker, modified BACs are identified by specific PCR amplification of the mutated BAC from cultures of pooled bacterial cells. Each pool represents about 10 electroporated cells from the original recombination mixture. Subsequently, individual clones containing the desired alteration are identified from the positive pools. Using this BAC modification method, we have observed a frequency of one recombinant clone per 90–260 electroporated cells. The combination of high targeting frequency and the sensitive yet selective PCR‐based screening method makes BAC manipulation using oligonucleotides both rapid and simple. genesis 29:14–21, 2001. Published 2001 Wiley‐Liss, Inc.

Identification and analysis of recombineering functions from Gram-negative and Gram-positive bacteria and their phages.

We report the identification and functional analysis of nine genes from Gram-positive and Gram-negative bacteria and their phages that are similar to lambda (λ) bet or Escherichia coli recT. Beta and RecT are single-strand DNA annealing proteins, referred to here as recombinases. Each of the nine other genes when expressed in E. coli carries out oligonucleotide-mediated recombination. To our knowledge, this is the first study showing single-strand recombinase activity from diverse bacteria. Similar to bet and recT, most of these other recombinases were found to be associated with putative exonuclease genes. Beta and RecT in conjunction with their cognate exonucleases carry out recombination of linear double-strand DNA. Among four of these foreign recombinase/exonuclease pairs tested for recombination with double-strand DNA, three had activity, albeit barely detectable. Thus, although these recombinases can function in E. coli to catalyze oligonucleotide recombination, the double-strand DNA recombination activities with their exonuclease partners were inefficient. This study also demonstrated that Gam, by inhibiting host RecBCD nuclease activity, helps to improve the efficiency of λ Red-mediated recombination with linear double-strand DNA, but Gam is not absolutely essential. Thus, in other bacterial species where Gam analogs have not been identified, double-strand DNA recombination may still work in the absence of a Gam-like function. We anticipate that at least some of the recombineering systems studied here will potentiate oligonucleotide and double-strand DNA-mediated recombineering in their native or related bacteria.