33be detected automatically using a simple thresholding method.
We made two different chromosomal yfp-mutL constructs.
In one case, we inserted yfp-mutL at the position of the native
lacZ such that yfp-mutL is expressed from the inducible lac
promoter (Plac). In the other case, we inserted yfp-mutL at the
site of native mutL gene. In this case yfp-mutL is constitutively
expressed from the native mutL promoter (PmutL). These two
constructs are suitable for different applications. Working with
fluorescently tagged proteins expressed at their physiological
levels might seem preferable as it prevents the toxicity and the
nonspecificity of the overproduction of the fluorescently
labeled proteins. However, we found that the overexpression
of the fluorescent MutL is not toxic and that it does not affect
the frequency of MutL foci. In addition, the PmutL is a weak
promoter, leading to the synthesis of on average 113 MutL
dimers per E. coli cell [ 11 ], which in the case of the yfp-mutL
means a low MutL fluorescence. Detection of the low fluores-
cence requires high excitation light intensity or long exposure
of cells to excitation light, which causes the bleaching of fluo-
rescence, leading to the signal loss and underestimations (see
Note 1). Expression of the yfp-mutL from the Plac promoter
is higher compared to the expression of the yfp-mutL from the
PmutL. Therefore, detecting fluorescent MutL foci using this
construct requires less illumination.
The expression of the yfp-mutL from the Plac promoter
varies depending on the growth medium. In some media, leaky
expression from the Plac will produce enough fluorescent
MutL allowing for the complete complementation of the
native mutL inactivation. In others, the Plac inducer, IPTG,
should be added to the growth medium to assure the expres-
sion of sufficient amount of fluorescent MutL. It is important
to determine before starting a new experiment if IPTG should
be supplied to the growth medium or not (see Note 2).
Expression level of the yfp-mutL gene should be sufficient to
restore wild-type mutation rate to the strain whose native
chromosomal mutL gene is deleted. This can be done quanti-
tatively or qualitatively (see Note 3) by a classical mutagenesis
experiments. For the quantitative test, grow the strain deleted
for the native chromosomal mutL gene, which expresses the
fluorescent MutL overnight in a desired medium supple-
mented or not with IPTG. Do the same for the reference wild-
type E. coli strain. Upon growth to saturation, dilute 10^7 -fold
the saturated cultures to eliminate preexisting rifampicin-resis-
tant (RifR) mutants and grow them again to saturation. Plate
the dilutions of the saturated cultures on the selective medium
plates, which contains LB supplemented with 100 μg/mL
rifampicin to select RifR mutants, and on the LB medium plates
to determine the total number of colony forming units.
Detecting Mutations in Living Cells