32
2 Materials
- E. coli MG1655 expressing the xfp-mutL gene and deleted for
the native chromosomal nonfluorescent mutL gene. The dele-
tion of the native mutL gene prevents the recruitment of the
nonfluorescent MutL protein to the sites of DNA mutations.
This is important for the proper visualization of DNA muta-
tions as the recruitment of the nonfluorescent MutL to such
DNA sites could decrease the fluorescent signal of MutL foci
or obstruct it completely. Different fluorescent proteins can be
used to render the MutL protein fluorescent. We constructed
the fusions of genes coding for enhanced green fluorescent
protein (egfp), yellow fluorescent protein (yfp), or cyan fluores-
cent protein (cfp) to the mutL gene. We used these different
fluorescent constructs in different experimental setups depend-
ing on our need to visualize only the fluorescent MutL, to
investigate the colocalization of different MMR proteins [ 7 ,
10 ], or to investigate the colocalization of the fluorescent
MutL with the replisome or different regions of the E. coli
chromosome. xfp- mutL gene was either cloned on a plasmid or
inserted into E. coli chromosome. Chromosomal construction
is preferred because it produces less cell-to-cell heterogeneity
in the amount of the cytoplasmic fluorescent MutL. Reduction
of such noise makes the data analysis easier. When cell-to-cell
variations in cytoplasmic fluorescence are limited, the foci can
2.1 Escherichia coli
Strains
Fig. 1 Fluorescent MutL protein tags the emerging mutations. (a) E. coli wild-type cells growing on agarose
pad and expressing the fluorescent MutL. All cells show uniform cytoplasmic fluorescence. The bright fluores-
cent MutL spot (indicated by an arrow) tagging emerging mutation is visible in only one cell. (b) Population of
the mutH cells, which are MMR deficient mutants, produce 50–100-fold more mutations and produce more
MutL fluorescent foci per cell, than wild-type cells
Marina Elez et al.