37
type strain, overnight in a desired medium, supplemented or
not with IPTG. Upon growth to saturation, plate 20 μL of the
saturated cultures on the LB plates containing 100 μg/mL
rifampicin to select RifR mutants and incubate the plates for
24 h at 37 °C. If fluorescent MutL is sufficiently expressed to
complement native mutL gene inactivation, a small number
(<5) of RifR colonies will grow per spot, which is comparable to
the wild-type strain. In contrast, if there are significantly (50–
100-fold) more colonies per spot than for the wild-type strain,
native mutL gene inactivation is not fully complemented.
- For wild-type E. coli cells, the expected frequency of RifR
mutants is around 2 × 10 −^8. Cells inactivated for MMR (mutS,
mutL, mutH, uvrD) show 50–100-fold higher frequency of
RifR mutants.
- In the case of the Plac construct, the insertion of the yfp-mutL-
cam in the chromosome leads to the deletion of lacZ gene.
Thus, white, blue screen can be used to search for positive
clones amongst the selected chloramphenicol resistant ones.
- In this case, make sure that the native chromosomal mutL is
deleted, not simply inactivated, before transforming the cells
with the DNA coding for the yfp-mutL-cam. Otherwise, the
yfp-mutL-cam DNA inserts incompletely in the native chro-
mosomal mutL site on the E. coli chromosome.
- If you wish to compare the results obtained in different experi-
ments, pay attention to O.D, because the mutation rate could
vary at different O.D.s. Our preliminary results indicate that
mutation rates might be lower at the entry into stationary
phase compared to mid- and early-exponential growth phase.
- It is important to dilute the saturated culture at least 400-fold.
This allows the majority of cells to exit the stationary phase
before imaging. Some stationary phase cells accumulate occa-
sionally “nonfunctional” MutL aggregates, which are not tag-
ging DNA mutations. These foci, contrary to “functional”
fluorescent MutL foci, form independently of the MutS pro-
tein. Consequently, they are detectable in the stationary phase
mutS cells. Diluting enough helps getting rid of such nonspe-
cific MutL aggregates.
- Make sure that the growth medium has been filtered and that
agarose has been completely dissolved. This will help to
decrease the autofluorescence of the agarose pad.
- In this case, make sure not to leave the cells on the slide for
more than 20 min before imaging. Due to nutrient lack, cells
might enter the stationary phase and cease replicating. In these
conditions cells mutate less and “nonfunctional” mutL aggre-
gates start appearing (see Note 8).
Detecting Mutations in Living Cells
