Figure 7. Schematic representation of λ phage constructs used for recombineering.

(a) The λ prophage with some of its genes is shown integrated in the bacterial chromosome. It is adjacent to the biotin genes, bioAB. Bacterial DNA is shown as a blue bar; phage DNA is a red bar; and a region containing a deletion and/or a substitution is a white bar. The complete λ prophage is flanked by its attachments site att, where integration occurred. The int and xis genes located in the pL operon encode functions to integrate and excise the phage DNA into and out of the bacterial chromosome. Two operons with their promoters pL and pR are indicated. Transcription of the promoters is controlled by the λ CI857 repressor. This repressor is temperature sensitive in that it is active and represses the promoter at 30–32°C but is inactive at 42°C allowing transcription. The N functions as an anti-transcription terminator and prevents RNA polymerase termination of pL transcripts at terminators tL1, tL2, and tL3. The red genes exo, bet, and gam encoding the homologous recombination functions are also in the pL operon. The kil gene is adjacent to gam and when expressed for over 1 hr kills the bacterial cell ³². The replication genes O and P are in the pR operon. Cro functions as a partial repressor of the pL and pR operons when CI is inactive at 42°C. The lysis and structural genes, SRA-J, are shown located beyond their regulator Q. The λ TetR phage used for recombineering is identical to the above complete prophage with the exception of five changes. The replication gene P has an amber mutation, the cro gene also has an amber mutation, there is an added mutation ind1 in the cI857 repressor gene, and the tetA gene encoding tetracycline resistance replaces rex. (b) The defective prophages in DY380, SW102, and the HME strains have the lytic pR operon deleted from cro through the bioA genes as shown in brackets. The right att site is also deleted preventing any excision of this prophage. In DY380 and SW102 cro through bioA is replaced by the tetracycline resistance cassette, tetRA. (c) The mini-λ phage DNA is shown with the lytic genes cro through J deleted. In different constructs of the mini λ, the cro-J region is replaced with various drug resistant cassettes. Mini-λ has both att sites plus int and xis, which allows for its integration and excision. (d) The λ segment on the pSIM plasmids is shown. The plasmid backbone is not shown. The genes from cro to att are removed as well as genes beyond tL3 including int and xis. The red genes are connected directly to pL by a deletion that removes kil through N. The drug resistant marker characteristic of each SIM plasmid replaces the rex gene adjacent to cI857. The basic features are conserved in all of these Red expression constructs. Red is left under the native phage controlling elements for optimal expression and regulation. The pL promoter drives gene expression and is controlled by the temperature sensitive but renaturable λ CI857 repressor. In all, the cro gene is inactive to maximize pL expression, and the replication genes are absent or inactive to prevent lethal effects on the cell.