- SDS-PAGE sample buffer: 0.09 M Tris–HCl pH 6.8, 20%
(vol/vol) glycerol, 2% (wt/vol) sodium dodecyl sulfate
(SDS), 0.02% (wt/vol) bromophenol blue, 0.1 M dithiothrei-
tol (DTT). - Donor strain: This is the strain that contains the transposon (see
Notes 1and 2 ) that has to be mobilized into your recipient
strain of interest (seeNote 3). We will use theE. colistrain
BW20767 carrying the suicide vector, pUT mTn5lux kan2,as
an example. - Recipient strain: This is the strain that you wish to mutagenize.
We will use theEnterobacter cloacaestrain JLD401 as an exam-
ple (seeNote 4). - 100 and 250 ml Erlenmeyer flasks.
- Shaking incubator.
- Refrigerated centrifuge capable of 10,000g.
- Sonicator.
- 1.5 and 15 ml plastic tubes.
- 96-Well microtiter plates.
- Sterile toothpicks or pipet tips.
- Spectrophotometer/luminometer.
- Sequencing and PCR primers.
3 Methods
3.1 In Silico
Analyses for
Identification of a LuxR
Solo/Orphan
3.1.1 Identifying a LuxR
Solo
Identification of a LuxR solo (seeNote 5) involves first determining
if the protein has sequence features typical of a QS LuxR protein
and then ascertaining the characteristics of the genome hosting this
protein to find out whether it is part of a LuxI/LuxR QS system or
occurs alone (seeNote 6). In case of metagenome sequences these
analyses might be limited if a complete genome sequence is not
available. In that case, a particular contig covering several 100 Kbs
might be used as the template for analyses of the context of the
LuxR-coding region. The details for use of each bioinformatic tool
mentioned here are available in the respective web sites and they
should be referred to for further information.- Identification of QS LuxR proteins: Go to the InterProScan
web page. Enter the sequence/s of interest (either protein or
DNA) in the search box or upload from a file. Choose the
sequence type as either protein or DNA and if latter choose
the translation table. Enter an e-mail address and submit job to
begin the search for protein signatures of the sequence. In the
results page, identify protein sequences showing IPR005143
with the signature “transcriptional factors LuxR-like,
Solo/Orphan QS Receptors 149