Expression, Purification, and Functional Analysis of Novel RelE Operon from X. nematophila

Figure 1

Genetic organization of toxin-antitoxin (TA) module of Xn-relE operon. Xn-relE TA operon is located on complementary strand in the genome of X. nematophila. Primers position and orientation for cloning of different domains depicted with arrows. Overlapping sequence of 11 nucleotide base pair is shown between the two modules. For cloning ORF 2 (285 bp) encoding Xn-relE toxin gene in pGEX4T expression vector, primer pairs 4 and 6 with BamHI site at 5′ end and XhoI site at 3′ end, respectively, were used. Primer pairs 1 and 5 with BamHI at 5′ and XhoI at 3′ end were used for directional cloning of antitoxin gene (252 bp) in pET28(a). Primer 2 with PstI site at 5′ end and primer 7 with HindIII site at 3′ end were used in cloning ORF2 (285 bp) encoding toxin Xn-relE gene in pBAD. Complete TA operon (526 bp) containing antitoxin gene (ORF1) followed by toxin gene (ORF2) was amplified and cloned in pBAD using primer 3 with PstI site at its 5′ end and primer 7 with HindIII site at its 3′ end.