and rearrange quite frequently. Find a new source of the vector.
Another issue is that the RP4 conjugative apparatus must be
able to initiate conjugation with your recipient strain. The RP4
apparatus does exhibit phase variable host specificity, which can
be problematic. In this case, try other donor strains such as
SM10λpiror S17λpir[49]. If too many colonies are obtained,
repeat the mating and plate dilutions of the mating mix onto
the selective plates until isolated colonies are obtained.- Assuming 4400 genes in the typical bacterial genome, and
given the fact that transposons can insert into reading frames
in two orientations, 8800 fusions are required to provide
roughly 1coverage of the genome. However, this does not
account for the Poisson distribution of insertions. When
accounting for the Poisson distribution, screening 8800
fusions will give 63% coverage (a 63% chance that any given
gene is screened at least once), screening 17,000 fusions will
give 86% coverage, and screening 27,000 fusions will give 95%
coverage. - It is helpful to know which growth conditions are permissive for
the LuxR solo in question. If the fusions are screened for AHL
responsiveness during growth in conditions in which the LuxR
of interest is not expressed, or not stable, etc., the AHL-
responsive fusions will test as nonresponsive. This is somewhat
of a chicken-and-egg problem as permissive conditions are not
usually known until a fusion can be obtained and tested. In this
case, best guesses based on the environment of the bacterium are
required. If no AHL-responsive fusions are obtained, one can
adopt an alternative screening strategy in which the LuxR solo is
overexpressed, in the hope that this provides activity under
nonideal conditions. For instance, we identified our firstsdiA-
dependent fusions inSalmonellausing overexpression because
we did not know the proper AHLs to use or the best growth
conditions [50]. Optimizing conditions with these fusions led to
later screens being performed without overexpression and using
what was an optimal condition for SdiA ofSalmonellagrowth in
motility agar [51, 52]. However, this optimal condition did not
turn out to be optimal forE. coli[53].
References
- Fuqua C, Parsek MR, Greenberg EP (2001)
Regulation of gene expression by cell-to-cell
communication: acyl-homoserine lactone quo-
rum sensing. Annu Rev Genet 35:439–468 - Waters CM, Bassler BL (2005) Quorum sens-
ing: cell-to-cell communication in bacteria.
Annu Rev Cell Dev Biol 21:319–346
3. Whitehead NA, Barnard AM, Slater H et al
(2001) Quorum-sensing in gram-negative bac-
teria. FEMS Microbiol Rev 25:365–404
4. Case RJ, Labbate M, Kjelleberg S (2008)
AHL-driven quorum-sensing circuits: their fre-
quency and function among the Proteobac-
teria. ISME J 2:345–349
Solo/Orphan QS Receptors 157