we describe a simple method to experimentally confirm ligand
binding specificities of the LuxR solo protein. Finally, a method
that can be used to identify possible gene targets of LuxR solos is
described.2 Materials
2.1 Hardware and
Software for In Silico
Identification of LuxR
Solos/Orphans
- A personal computer connected to the Internet.
- Complete genome sequences available at public databases like
NCBI, GeneDB (http://www.genedb.org/), and TIGR
(http://www.tigr.org/) or genome sequences of strains of
interest likePseudomonasGenome Database (http://www.pseu
domonas.com/), EcoCyc (http://ecocyc.org/), or sequences
obtained from samples of interest. - The InterPro database freely available athttp://www.ebi.ac.
uk/interpro. - SynTax:http://archaea.u-psud.fr/SyntTax.
- FGENESB: http://www.softberry.com/berry.phtml?topic¼
fgenesb&group¼programs&subgroup¼gfindb. - Clustal W:http://embnet.vital-it.ch/software/ClustalW.html.
- Clustal Omega:https://www.ebi.ac.uk/Tools/msa/clustalo/.
- BPROM: http://www.softberry.com/berry.phtml?topic¼
bprom&group¼programs&subgroup¼gfindb. - MEME Suite:http://meme-suite.org/.
2.2 Studies on the
Ligand of the LuxR
Solo/Orphan
- Plasmids pQE30, pQE31 and pQE32.
- Structurally different AHLs.
3.E. coliM15 (pREP-4). - Antibiotic stock solutions: 100 mg/ml ampicillin (Ap),
25 mg/ml kanamycin (Km), and 25 mg/ml nalidixic acid
(Nal) dissolved in distilled water and filter sterilized. A few
drops of 10 M NaOH are required to dissolve Nal in distilled
water before sterilization by filtration. - Lysogeny broth (LB): 1% (wt/vol) bacto-tryptone, 0.5%
(wt/vol) yeast extract, 1% (wt/vol) sodium chloride in distilled
water. Sterilize by autoclaving. - LB agar: LB plus 1.5% (wt/vol) agar. Sterilize by autoclaving.
- 1 M Isopropyl-β-D-thiogalactoside (IPTG) dissolved in dis-
tilled water. - Binding/lysis buffer: 50 mM NaH 2 PO 4 , 300 mM NaCl,
10 mM imidazole; adjusted to pH 8.0 using NaOH.
148 Vittorio Venturi et al.