- Centrifuge the lysate at 10,000gat 4C for 30 min. Take
and save the cleared supernatant: it represents the soluble
fraction. Resuspend the pellet in 5 ml of binding/lysis buffer:
this, instead, represents the insoluble fraction. - Analyze the samples representing soluble and insoluble pro-
teins in SDS-PAGE as follows: add 5μl of SDS-PAGE sample
buffer to 5μl of the two protein extracts. Microcentrifuge at
maximum speed for 2 min and load each sample on a 12% SDS-
PAGE gel. Run the gel at 200 V for ~45 min and determine
whether the overexpressed QS-LuxR solo is located in the
soluble or insoluble fraction by visual inspection of the gel
after Coomassie staining (more details on SDS-PAGE analysis
can be found in Chapter6 of this book).
3.3 Identifying LuxR
Solo Regulon Members
Using Random
Transcriptional
Fusions
Once a LuxR solo has been identified, the regulon can be identified
by either screening random transcriptional fusions or using RNA-
seq and/or ChIPseq. RNAseq has the advantage of more complete
coverage of the genome, but the genes that appear to be regulated
by the LuxR solo must be confirmed at a later time, either with
qRT-PCR or with transcriptional fusions. The advantage to screen-
ing random transcriptional fusions is that the resulting fusions can
be used for confirmation and further studies immediately. In this
chapter we describe the screening of random transcriptional
fusions, while RNAseq and ChIPseq are the topics of other reviews;
the experiment described will highlight genes that respond to AHL
(s) which have to be confirmed as belonging to the LuxRsolo
regulon by further analysis as checking their response to AHLs in
the LuxRsolo mutant.3.3.1 Generation
of Fusions
- Grow 5 ml overnight cultures of the donor strain (E. coli
BW20767 carrying pUT mTn5lux kan2) and your recipient
strain of choice (for example,E. cloacaeJLD401) at 37C with
shaking. In this case, both cultures can utilize LB broth as
growth medium. The donor culture should include 100μg/
ml Km and the recipient culture should include 25μg/ml Nal
to insure that those markers are maintained. - The following day, remove the antibiotics from the cultures. To
do this, centrifuge the 5 ml of both cultures for 10 min at
10,000gto achieve pelleting of the bacteria. Remove the
supernatant from each pellet and resuspend the pellet in 5 ml
fresh LB. - To begin the mating of the strains, mix 100μl of the donor and
the recipient together in a microcentrifuge tube (final volume
200 μl); set up between 10 and 50 matings. Spot the entire
volume of each tube, 200μl, on an LB agar plate and incubate
overnight at 37C. Having each plate represent a separate
mating insures that mutants obtained from separate plates are
Solo/Orphan QS Receptors 153