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. Author manuscript; available in PMC: 2013 Jan 1.
Published in final edited form as: Mol Microbiol. 2011 Nov 22;83(1):96–109. doi: 10.1111/j.1365-2958.2011.07915.x

Figure 1.

Figure 1

Deletion of the lgt gene and in situ complementation. (A) lgt was removed by Cre-loxP system. Underlined region was amplified by PCR to confirm deletion as shown in (B). (B) Ethidium bromide stained agarose gel showing PCR analysis of lgt depletion. Primers were designed to amplify the internal portion between BA5392 and BA5390. W: wild-type B. anthracis, lgt: lgt mutant. (C) For in site complementation, the pSC plasmid containing BA5392-BA5389 was introduced into the lgt knockout mutant, which was then grown at the restrictive temperature. The single-crossover insertion event was selected by the EmR phenotype. Removal of the EmR along with the pSC backbone from the chromosome was achieved by Cre-mediated recombination following transformation with pCrePAS. Growth at 37°C eliminated the pSC and pCrePAS plasmids, resulting in the (re)insertion of BA5391 (lgt gene). Underlined regions were amplified by PCR to analyze the resulting strain as shown in (D). (D) Ethidium bromide stained agarose gel showing PCR analysis of the complementation of lgt gene (BA5391) and the duplication of BA5392. W: wild-type B. anthracis, lgt: lgt mutant, compl: in situ complemented mutant.