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Chapter 19 : Recombineering in Prokaryotes

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Abstract:

This chapter describes the present state of recombineering in and details the essential elements of the system. The success of this technology has encouraged its development for use in other organisms. The chapter discusses that goal and some obstacles that must be overcome in order to fully utilize recombineering in other prokaryotes. It emphasizes on the biochemistry of the proteins used for recombineering, the genetic manipulations possible, and the relative efficiencies of each. Several different types of genetic construction can be engineered by recombineering, and the desired genetic product dictates the type of DNA substrate used in the recombineering reaction. The following four types of substrates have been used successfully in laboratories: PCR products; short, partially dsDNA created by annealing ss-oligos; gapped linear plasmid DNA; and ssDNA oligonucleotides. Exo and Beta are needed to process the PCR product prior to its incorporation into the chromosome, and Gam is needed to prevent the degradation of the linear dsDNA by the RecBCD nuclease and possibly by the SbcCD nuclease. Recombineering in more distantly related bacteria will be facilitated by the identification and characterization of Red-like functions in phages from those organisms. Other requirements for the ready use of recombineering are those in common with other modes of genetic analysis, such as the development of systems permitting regulated gene expression and means of easily introducing DNA into the organism under study.

Citation: Thomason L, Costantino N, Sawitzke J, Datta S, Bubunenko M, Court D, Myers R, Oppenheim A. 2005. Recombineering in Prokaryotes, p 383-399. In Waldor M, Friedman D, Adhya S (ed), Phages. ASM Press, Washington, DC. doi: 10.1128/9781555816506.ch19

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Tables

Generic image for table
TABLE 1

Typical recombination efficiencies for standard recombineering reactions

DY330, Δ() gal490 (λI857-bioA) ( ).

DY411, W3110 (λI) < > with 34 bp of deleted ( ).

DY378, W3110 (λI) ( ).

HME6, W3110 Δ()I).The oligo creates a T-C mismatch that is well repaired by the MMR system ( ).

Citation: Thomason L, Costantino N, Sawitzke J, Datta S, Bubunenko M, Court D, Myers R, Oppenheim A. 2005. Recombineering in Prokaryotes, p 383-399. In Waldor M, Friedman D, Adhya S (ed), Phages. ASM Press, Washington, DC. doi: 10.1128/9781555816506.ch19
Generic image for table
TABLE 2

Recombination efficiencies of various recombineering systems with ss-oligos and dsDNA

W3110, K-12 IN().

TS616, serovar Typhimurium LT2 ::Tn from Genetic Stock Center.

NA, not applicable.

From reference .

Minimal prophage on plasmid (see text and Color Plate 19).The data in the table were generated with the Cm plasmid.

From reference .

ND, not determined.

From reference .

From reference .

10 mM arabinose.

1 mM arabinose.

Citation: Thomason L, Costantino N, Sawitzke J, Datta S, Bubunenko M, Court D, Myers R, Oppenheim A. 2005. Recombineering in Prokaryotes, p 383-399. In Waldor M, Friedman D, Adhya S (ed), Phages. ASM Press, Washington, DC. doi: 10.1128/9781555816506.ch19

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