Lambda placMu: a transposable derivative of bacteriophage lambda for creating lacZ protein fusions in a single step

Abstract

We isolated a plaque-forming derivative of phage X, A placMul, that contains sequences from bacteriophage Mu enabling it to integrate into the Escherichia coli chromosome by means of the Mu transposition system. The Mu DNA carried by this phage includes both attachment sites as well as the cI, ner (cII), and A genes. X placMul also contains the lacZ gene, deleted for its transcription and translation initiation signals, and the lacY gene of E. coli, positioned next to the terminal 117 base pairs from the S end of Mu. Because this terminal Mu sequence is an open reading frame fused in frame to lacZ, the phage can create lacZ protein fusions in a single step when it integrates into a target gene in the proper orientation and reading frame. To demonstrate the use of this phage, we isolated lacZ fusions to the malB locus. These showed the phenotypes and regulation expected for malB fusions and could be used to isolate specialized transducing phages carrying the entire gene fusion as well as an adjacent gene (malE). They were found to be genetically stable and rarely (<10-7) gave rise to secondary Lac' insertions. We also isolated insertions into high-copy-number plasmids. The physical structure of these phage-plasmid hybrids was that expected from a Mu-dependent insertion event, with the A placMu prophage flanked by the Mu attachment sites. Lac' insertions into a cloned recA gene were found at numerous positions and produced hybrid proteins whose sizes were correlated with the position of the fusions in recA. Gene fusions to the Escherichia coli lacZ gene (encoding

Arabinose-Induction of lac-Derived Promoter Systems for Penicillin Acylase Production in Escherichia coli

“…PAC was assayed at 37°C using penicillin G as a substrate (Gang and Shaikh, 1976). The amount of enzymatic reaction (Bremer et al, 1984) MD∆P7 A penicillin-sensitive and 6-APA-resistant strain derived from HB101 this lab (Chou et al, 1999a) plasmid pTrcKnPAC2902 Ptrc::pac, Ori (pBR322), Kn R this lab (Chou et al, 1999b) pTrcKn29S Ptrc::LL pac, Ori (pBR322), Kn R this lab (Xu et al, 2005) pETKn29a PT7::pac, Ori (pBR322), Kn R this lab (Xu et al, 2006) pARDegP ParaB::degP, Ori (pACYC184), Cm R this lab (Pan et al, 2003) product of 6-aminopenicillanic acid (6-APA) was quantified using a colorimetric method developed previously (Balasingham et al, 1972). All assays were conducted in duplicate.…”

Characterization of the Salmonella typhimurium mgl operon and its gene products

“…Isolation of mgl-lacZ fusions in vivo. mgl-lacZ protein fusions were isolated in strain MC4100 carrying the mgl hybrid plasmid pHG4 by using phage XplacMu3 as described 38 MULLER, HEINE, AND BOOS (9). To 0.1 ml of logarithmically grown cells resuspended in 10 mM MgSO4, 50 p.l of XplacMu3 (4 x 109 phages per ml) and 50 pul of the helper phage XpMu5O7.3 (4 x 109 phages per ml) were added.…”

Regulation of the Escherichia coli uncH gene by mRNA secondary structure and translational coupling

“…Plasmids lacZ fusions to uncH were originally created using ?.p/acMu3 {Bremer et al. 1984) to construct in-frame uncH'-'lacZ\us\on genes in plasmid pRPG51 (Gunsalus et at.. 1982).…”