A set of recombineering plasmids for gram-negative bacteria
Introduction
Recombineering is a new means of in vivo genetic engineering that allows DNA modifications to be made easily and efficiently. It is a highly effective method for functional genomic analysis, engineering a wide variety of DNA rearrangements and combining genes with special genetic elements or tags (Copeland et al., 2001, Court et al., 2002). Recombineering allows a researcher to carry out DNA modifications and cloning without restriction enzymes or DNA ligases (Yu et al., 2000, Copeland et al., 2001, Court et al., 2002). The modifying DNA for recombineering is either a double-stranded (ds) PCR product (Murphy et al., 2000, Yu et al., 2000, Lee et al., 2001, Court et al., 2002) or a single-stranded oligonucleotide (oligo) (Ellis et al., 2001, Swaminathan et al., 2001) carrying short regions of target homology at the ends which can be precisely recombined in vivo with its substrate sequences onto any episome within the cell. Recombination between the short homologies is catalyzed by the λ Red functions, Exo, Beta and Gam. The λ Gam protein prevents degradation of transformed linear dsDNA by the host RecBCD and SbcCD nucleases (Unger and Clark, 1972, Kulkarni and Stahl, 1989) while Exo resects the 5′ ends of the dsDNA (Little, 1967) to generate 3′ ssDNA overhangs. Beta binds to these ssDNA overhangs, as well as to oligos, ultimately pairing them with a complementary ssDNA target (Karakousis et al., 1998, Li et al., 1998).
Our laboratory uses a defective λ prophage for optimal expression of the Red functions in E. coli (Yu et al., 2000, Ellis et al., 2001). This defective prophage contains the phage immunity region and the main leftward operon under control of the pL promoter (Fig. 1). The rightward operon encoding the DNA replication genes, lysis cassette, and the structural genes has been removed by a deletion that extends from cro through the right attachment site, attR and into the bacterial biotin gene bioA. The exo, bet and gam genes are expressed from the pL operon under the control of the temperature-sensitive (ts) repressor, CI857. At low temperature (30–34 °C) the repressor remains active and blocks the pL promoter, shutting off transcription of the red genes. A brief temperature shift to 42 °C results in a transient denaturation of the repressor, allowing Red expression. On shifting back to low temperature the repressor renatures, binds to pL, and again turns off the Red system. Following heat inactivation of the repressor, the expression of gam, bet and exo are initially prevented by the transcription terminators present between pL and the red genes unless the N anti-termination function modifies RNA polymerase to prevent transcription termination (Gottesman et al., 1980).
Here we report a modification of the prophage strain and derive from it a set of plasmids carrying a minimal Red expression cassette under endogenous λ repressor control. These new vectors can be introduced by transformation to different bacterial backgrounds, and in some of them, a ts replication defect provides a means for easy curing from the host. We also report two recombineering applications that are useful for plasmid engineering in general: 1) a technique to clone any gene with a linked drug marker to a multicopy vector by gap-repair and 2) a means to alter the origin of replication (ori) of a plasmid, hence changing its copy number and host specificities.
Section snippets
Bacterial strains
Unless otherwise specified, strain construction was done using recombineering technology (Yu et al., 2000, Ellis et al., 2001). Strain DY378 is W3110 {λ cI857Δ(cro-bioA)} (Yu et al., 2000). DY406 was constructed by replacing kil to sieB of λ nucleotide (nt) 33246–35015 (Daniels et al., 1983) in DY378 with a cassette containing both the chloramphenicol (cat) and sacB genes (Lee et al., 2001). DY432 was constructed from DY406 by replacing the cat-sacB cassette plus adjacent λ DNA sequence using
Construction of a minimal Red expression prophage
The minimal prophage was created by removal of the λ N through kil genes in the pL operon and replacement of rexA and rexB by a drug resistance cassette, either cat or amp (see Section 2.1). The N through kil deletion removes all transcription terminators between pL and the Red genes and makes Red expression N-independent. In this minimal construct, pL is regulated by CI857 with operators OL and OR ensuring tight repressor control. Raising the temperature to 42 °C inactivates the repressor and
Discussion
Here we report the construction of a series of Red expression plasmids, retaining the native λ control, but with a wider host range. Recombineering techniques were used to create these vectors by in vivo plasmid cloning. We utilized the λ Red system to both gap-repair a DNA segment of choice onto a vector and exchange the plasmid origin to alter copy number and/or host range.
The plasmids constructed in this study coordinately express gam, bet and exo of λ in their natural prophage context under
Acknowledgements
We thank L. Thomason, J. Sawitzke, A. Oppenheim, and M. Bubunenko for the many discussions and help with the manuscript. We are grateful to the Helinski laboratory for providing the plasmid pRR10-ts97 and to Eric Miller for the pBBR1-derived plasmid. This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer and in part by a Trans NIH/FDA Intramural Biodefense Program Grant from NIAID to DLC.
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