Part:BBa_K2229200

OmpR234 Expressing Construct

An OmpR234-based construct that employs the strong promoter/strong RBS (K880005) combination to up-regulate expression of OmpR234, which in turn activates the csgD promoter to upregulate production of curli fibers in Escherichia Coli.

Construct Design

This construct was built to upregulate curli production by overexpressing OmpR234. We acquired all parts from the iGEM distribution kit: a strong promoter and strong RBS combination (BBa_K880005) to maximize protein production, strong RBS (BBa_B0034), ompR234 (BBa_K342003), and a double terminator (BBa_B0015) to end transcription.

For strong OmpR234 expression (BBa_K2229200), ompR234 was inserted before BBa_B0015 (BBa_S05398) and then behind BBa_K880005.

PCR Check Gel

PCR Check for BBa_K2229200 using the forward and reverse primers VF2 and VR. The expected size of BBa_K2229200 (OmpR234 full construct) is 1200 bp (blue box).

Characterization

SDS-PAGE

BBa_K2229200 contains and expresses OmpR234 (BBa_K342003). SDS-PAGE results show OmpR234 protein around 27 kDa, which matches the expected size (Brombacher et al. 2006; Martinez & Stock 1997). This was compared to BBa_K342003, the original part which only contains the ORF.

SDS-PAGE results show that BBa_K2229100, BBa_2229200, and BBa_K2229300 overexpress CsgD, OmpR234, or both proteins, respectively. Predicted proteins from the curli operons are listed on the right, and E. coli expressing GFP was used as a positive control.

CONGO RED ASSAY

We used Congo Red (CR), a dye commonly used to measure biofilm production (Reinke & Gestwicki 2011). CR solution mixed with bacterial liquid cultures were transferred to 12-well plates with glass coverslips, and incubated at 37˚C for one day. If biofilms were present, the solution would appear red, which could be quantified by an absorbance value at 500 nm. Overexpression of OmpR234 (BBa_K2229200) in our experiments leads to about 8 times more biofilm compared to the control BBa_K342003.

Overexpression of OmpR234 (BBa_K2229200) leads to ~8 times more biofilm production than control. A) Congo red assay stains biofilm (red). B) Stained biofilm is solubilized in ethanol. C) Absorbance is measured at 500 nm.

When bacteria expressing OmpR234 (BBa_K2229200) were plated in petri dishes with glass coverslips, biofilms appeared thicker compared to controls (BBa_K342003).

References

Reinke, A A, and J E Gestwicki. “Insight into amyloid structure using chemical probes.” Chemical biology & drug design., U.S. National Library of Medicine, June 2011, www.ncbi.nlm.nih.gov/pubmed/21457473.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 200
  
• 1000
  COMPATIBLE WITH RFC[1000]