pInvRecA Vector

Generalized P1 transduction

DNA transduction using the E. coli bacteriophage P1 is highly useful in generating mutant microbial strains for genomic and proteomic studies, and for moving genes from one strain to another. Phage P1-mediated transduction can be used only with wild-type (RecA+) E. coli strains. However, most E. coli cloning strains, including Lucigen's E. cloni® 10G, are RecA– to provide stability of cloned fragments.

The pInvRecA vector transiently converts RecA– to the RecA+ phenotype, enabling phage P1 transduction to be performed in any E. coli strain (Sektas, M., et. al. (1999) BioTechniques 27, 911). The promoterless and inverted RecA gene is located between two convergently oriented Flp (Flipase) recognition target sequences (FRT) and adjacent to four strong transcription terminators (rrnBT1) (Figure 1A). This design prevents recombination before RecA induction. The RecA+ function can then be induced with heat-inactivated chlortetracycline (cTc). cTc activates the tetA promoter, inducing synthesis of the yeast Flp recombinase, which then inverts the recA gene, resulting in RecA protein production. The conversion to RecA+ enables homologous recombination upon phage P1 infection (Figure 1B).

Following standard P1 transduction at 37°C, the RecA– phenotype is completely restored by growing cells at 42°C. pInvRecA contains the selectable ts-replicon from pSC101 that allows for replication in E. coli at 30°C but not at temperatures above 37°C. The higher temperature incubation completely eliminates the pInvRecA vector from host cells.

Figure 1. The pInvRecA vector and its use.  Vector map:  RecA+  - promoterless recA gene; FRT - Flp yeast recombinase recognition sequence; FLP - Flp yeast recombinase gene; tetAp - tetracycline-resistance gene promoter; tetR - tetracycline resistance gene; ApR, ampicillin resistance gene; pSC101, pSC101 origin of replication; rep101Ts - temperature sensitive replication gene; rrnBT1 - transcription terminators. Vector use. Incubation with heat-inactivated chlortetracycline (cTc) activates the tetA promoter, resulting in expression of the FLP recombinase. FLP/FRT mediated inversion of the recA gene leads to production of RecA protein , which enables homologous recombination of DNA injected by P1-transducing phage.