34
Colonies should be scored after 24 h of incubation at
37 °C. Determine the average frequency of RifR mutants from
three to six independent experiments. The RifR mutant fre-
quency of the strain expressing fluorescent mutL and deleted
for the native chromosomal mutL gene should not be different
from that of the nonmodified wild-type reference strain (see
Note 4).
The yfp-mutL fusion that we designed and constructed
can be transferred into a desirable E. coli strain by a classical P1
transduction [ 12 ]. This is possible as we cloned the selectable
chloramphenicol resistant marker (cam) downstream of the
yfp- mutL gene (see Note 5). Otherwise, the DNA coding for
the yfp-mutL-cam can be amplified by PCR, using the plasmid
or the chromosomal templates, or synthesized de novo.
Obtained DNA fragments can be inserted into a desired posi-
tion on the E. coli chromosome by the Datsenko and Wanner
gene replacement method [ 13 ] (see Note 6).- E. coli strain expressing the mutL-yfp and deleted for the native
chromosomal mutL and mutS genes. To check that mutS is
properly deleted in the strain expressing fluorescent MutL and
inactivated for the native chromosomal mutL do the qualita-
tive Rif spot test as described above. Because MutS protein is
necessary for the MutL protein binding to emerging muta-
tions, this control strain allows distinguishing the “nonfunc-
tional” aggregates of MutL protein, which are MutS
independent, from the “functional” MutL foci tagging emerg-
ing mutations, which are MutS dependent. Therefore, the
presence of fluorescent foci in this strain indicates that the cul-
ture conditions lead to nonfunctional fluorescent MutL aggre-
gates and are consequently not suitable to detect mutations by
our method.
In principle, any growth medium can be used. We used the Plac
and the PmutL constructs grown in LB supplemented with
0.1 mM IPTG as well as in standard M9 minimal medium [ 12 ]
supplemented by 2 mM MgSO 4 , 0.003% vitamin B1, 0.001% ura-
cil, 0.2% casamino acids, and 0.01% glycerol. If Plac construct
should be used in a different growth medium check before starting
the experiment the fluorescent level of cells because too much
expression from the Plac inducible promoter in minimal media
complemented with pyruvate or glycerol could prevent accurate
detection of fluorescent MutL foci due to the high background
cytoplasmic fluorescence (see above). If not using the inducer,
check also that enough fluorescent MutL is produced in cells to
complement the inactivation of the native chromosomal mutL
gene (see above).2.2 Growth Medium
Marina Elez et al.