Part:BBa_K342003
OmpR234 protein, with higher effect on Curli promoter
This part is coding for a mutated version of the response regulator OmpR (J Bacteriol. 1998 180(9):2442-9). This protein will be phosphorylated by the associated-sensor EnvZ. This mutated phosphorylated protein is a better activator of the curli csgD promoter than the wild type OmpR. OmpR234 can activate the cryptic curli operon found in all known K12 strains and leads to the formation of thick biofilm on glass and polystyrene.
Usage and Biology
This part is widely spread in the world (see some example below) and can be used to induce a constitutive biofilm producing phenotype in bacteria. Microbiology. 2011 Jun;157(Pt 6):1640-50 Cornell, USA Appl Environ Microbiol. 2010 Oct;76(20):6939-41 Cornell, USA Appl Environ Microbiol. 2009 Dec;75(23):7426-35 Cornell, USA Water Res. 2008 Jun;42(12):3066-74, Columbia, USA Environ Sci Technol. 2007 Cornell, USA
Characterization (New data, 2011)
Microscopy tests
The pIG16 (BBa_J23119-OmpR234 in pSB1A2) and pIG3 (BBa_J23119 part in pSB1A2) were inserted in a strain constitutively expressing GFP, PHL1414 (MG1655 with a chromosomical insertion of GFP). The strains were respectively named S31 and S30.
In sterile empty plates, we introduced 10mL of M63G, with 100μL of Amp and 100μL of bacteria from a saturated liquid culture.
Then, we added 3 or 4 sterile glass slides and incubated at 30°C for 23 hours. If the strain produce some curli, it will form biofilm on the glass slides. We washed carefully the slides not to eliminate adherent bacteria.
We observed the surface of the slides with a fluorescent microscope to look if there was a biofilm. Several sites in the slides were photographed, the best pictures are presented here.
The complete protocol is available here
We can see that the strain containing the pIG16 plasmid is more adherent than the strain containing the control plasmid pIG3.
Conclusion: The BBa_J23119-OmpR234 part increases the stickiness of a part.
Quantitative adherence tests in plates
For characterization, the plasmids are introduced in PHL1414 strain. The strain with pIG16 plasmid is noted S31 and the control strain with the pIG3 plasmid is noted S30.
The effect of the plasmid on the adherence is measured.
To that end the strains are seeded in 24 wells plate, as described here
The measure of the OD 600 will give the percentage of adherence of the bacteria and the violet crystal coloration will give us a first idea on the formation of biofilm.
Conclusion: The OD600 is not significantly different for the two strains, which shows there is no effect of the plasmid on the growth.
The percentage of adherence is now measured for both strains
Conclusion: There is a significant increase (p=0.0008 in student t test) of stickiness when the part BBa_J23119-OmpR234 is introduced.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 137
- 1000COMPATIBLE WITH RFC[1000]
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