Part:BBa_K342003

OmpR234 protein, with higher effect on Curli promoter

This part is coding for a mutated protein OmpR. This protein will be phosphorylated by the complex EnvZ. This mutated phosphorylated protein is a better activator of the Curli promoter than OmpR.

Usage and Biology

This part can be used to induce a constitutive biofilm producing phenotype in bacteria.

Characterization

Microscopy tests

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The pIG16 and pIG3 were inserted in strain constitutively expressing GFP. 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 to eliminate no 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.

Quantitative adherence tests in plates

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For characterization, the plasmids are introduced in PHL1414 strain. The strain with pIG16 plasmid (18A-OmpR234 in pSB1A2) is noted S31 and the control strain with the pIG3 plasmid (18A part in pSB1A2) is noted S30.

The effect of the plasmid on the adherence is measured.

To that end the strains are seeded in 24 wells plate.

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 seemed to be the same for the two strains. There is no effect of the plasmid on the growth.

The percentage of adherence is now measured for both strains

Conclusion: we can see a strong increase of stickiness when the part 18A-OmpR234 is introduced.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 137
  
• 1000
  COMPATIBLE WITH RFC[1000]