Construction of the groEL1ΔC strain. (A) A schematic representation of the genomic organisation of groEL1 (Msmeg1583) in M. smegmatis mc2155 (1), the hygromycin-resistant groEL1ΔC mutant (2) and the unmarked groEL1ΔC deletion strain (3). Msmeg1582 is groES and Msmeg1581, 1584, 1585 and 1586 encode for proteins of unknown function. Zoom sequence of the C-terminal 40 amino acids of the groEL1 gene product, showing the histidine-rich C-terminal region. In the groEL1ΔC strain, the eleven C-terminal amino acids (white) were replaced by six different residues. HygR = hygromycin-resistance cassette. (B) Southern blot analysis performed with genomic DNA of M. smegmatis mc2155 (1), mc2155 carrying pJV53 (2), two correct hygromycin-resistant groEL1ΔC mutants (3-4) and two correct unmarked groEL1ΔC deletions (5-6). The genomic DNA was digested with EcoRI or SacI. The positions of the restriction sites EcoRI and SacI, relative to the start of groEL1, are presented as a full and dotted vertical line in A, respectively. The PCR product of primer pair Msmeg1583-F1.2 & Msmeg1583-R1 was used as a probe, shown as a dashed line under the genes in A1. (M) DNA marker VII, DIG labelled (Roche).
Noens et al. BMC Biotechnology 2011 11:27 doi:10.1186/1472-6750-11-27