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. Author manuscript; available in PMC: 2015 Jul 1.
Published in final edited form as: Methods Mol Biol. 2015;1276:13–29. doi: 10.1007/978-1-4939-2392-2_2

Fig. 2. Plasmids for rpoB and rpoC mutagenesis.

Fig. 2

mutant versions of shorter rpoA and rpoZ can be obtained synthetically from commercial sources. We introduce a desired substitution by site-directed mutagenesis (for example, QuikChange), adding a silent restriction site (if possible) for easy screening. We sequence the mutant fragment between two closest convenient restriction sites and then clone this fragment back into the original plasmid (to make sure there are no mutations elsewhere). Finally, we reclone a fragment flanked by two sites that are unique in pVS10 (or another similar vector).

A. Mutagenesis of the β subunit. We use three vectors that encode the N-terminally His6-tagged β subunit under control of Ptrc promoter most frequently. The wild-type plasmid pIA545 (shown)encodes the wild-type rpoB except a silent MfeI site at β residue 555 ; it complements a temperature sensitive defect in the rpoB gene. Two other plasmids have the same structure but lack the MfeI site; instead, they encode rifampicin-resistant rpoB alleles: βD516V in pIA223 and βS531F in pIA178. The activity of altered RNAPs can be monitored in the presence of rifampicin, to eliminate concerns of contamination with the wild-type core. Following mutagenesis, we reclone the NcoI-SbfI fragment, which encompasses almost entire rpoB)into a desired vector (e.g., pIA821).

B. Mutagenesis of the C-terminus of β and the N-terminal half of β′. pIA458 is a Litmus-based plasmid that does not encode any complete RNAP subunit. Mutagenized fragments can be transferred into a vector of choice (e.g., pIA900 for β′ mutants) on the SbfI-BsmI fragment. pIA458 has as an SpeI site in the intergenic region separating the rpoB and rpoC genes that can be used for screening .

C. Mutagenesis of the C-terminal half of the β subunit. We use pIA661, which encodes the wild-type rpoC except for a silent EagI site at β′ residue 675, most frequently. Two variants carrying antibiotic-resistant alleles, pIA388 and pIA635 (see Table 2) can be used instead. These plasmids encode the C-terminally His6 tagged β′ subunit under control of Ptrc promoter. The wild-type plasmid complements a temperature-sensitive defect in the rpoC gene. Following mutagenesis, we reclone the BsmI-HindIII fragment into a desired vector (e.g., pVS10).