Table 2.

Percentages of xylE-negative and xylE+ colonies found on second-crossover selection platesa of M. smegmatis FP101 with various rescue plasmids

M. smegmatis FP101 with the following plasmid(s)Antibiotic(s) used (same on both broth and plates)f% of yellow colonies% of white colonies
NoneKAN991
pCG76 and pMVHG1bKAN, STR, HYG991
pCG76:TBglfKAN, STR9010c
pMVHG1:Rv3808cKAN, HYG991
pMVHG1:Rv3808c and pCG76:TBglfKAN, STR, HYG0100d
pMVHG1:TBglf and pCG76:Rv3808cKAN, STR, HYG0100e
  • a The strains were grown in LB broth with the antibiotics indicated in the table and then plated on LB broth plates containing 10% sucrose and the antibiotics indicated here.

  • b This is a control for the effect of the empty pCG76 and pMVHG1 plasmids.

  • c Twelve of the white colonies were tested for the double-crossover event by Southern blot analysis after digestion of their DNAs with NruI, using the 1,595-bpglf-containing fragment as a probe. None of the colonies (data not presented) yielded the 1.23- and 2.41-kbglf-containing bands expected after a genuine (Fig. 5) second crossover event.

  • d Twenty-four of the white colonies were tested for the double-crossover event by Southern blot analysis after digestion of their DNAs with NruI followed by probing withglf. Thirteen yielded the 1.23- and 2.41-kbglf-containing bands expected for the genuine double-crossover strain. The Southern blot of three of these is presented in Fig. 5.

  • e All 17 colonies tested for the double-crossover event by Southern blot analysis after digestion of their DNAs with NruI using the 1,595-bpglf-containing fragment as the probe were found to be genuine double-crossover strains. The reason for the slightly different result here from that with the other pair of rescue plasmids (see footnote d) is unclear.

  • f STR, streptomycin; HYG, hygromycin.