Part:BBa_K2229300

Dual Expressing Construct of CsgD and OmpR234
The full construct places a strong Promoter/RBS (BBa_K880005) in front of csgD (BBa_K805015) and ompR234(BBa_K342003), transcriptional regulators of two curli operons, which control curli fiber production. Both csgD and ompR234 were acquired from the iGEM distribution kit. By expressing both proteins, we increase curli fiber production in Escherichia coli..

Construct Design

This construct was built to upregulate curli production by overexpressing CsgD and OmpR234 (a mutant form of OmpR which is constitutively active). 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), csgD (BBa_K805015), ompR234 (BBa_K342003), and a double terminator (BBa_B0015) to end transcription.

PCR Check Gel

PCR Check for BBa_K2229300 using the forward and reverse primers VF2 and VR. The expected PCR size of BBa_K2229300 (CsgD and OmpR234 expression) is 1900 bp (green box).

Characterization

Characterization and Improving Function of Previous Parts

Our new composite part BBa_K2229300 improves the function of two existing parts: BBa_K342003 (ompR234 ORF) and BBa_K805015 (csgD ORF).

Hypothesis

We hypothesized that biofilm production would be upregulated (in increasing order) if we overexpress CsgD, OmpR234, or both. Overexpression of CsgD would result in more curli monomers, but no transport proteins to carry the monomers out of the cell. Overexpression of OmpR234 would allow curli monomers to be exported and form fibers and biofilm. Finally, when both CsgD and OmpR234 are overexpressed, twice the amount of curli monomers should be made and exported to form fibers and biofilm.

SDS-PAGE

The original parts, BBa_K342003 (ompR234 ORF) and BBa_K805015 (csgD ORF), were cloned into expression devices, Bba_K2229200 and Bba_K2229100, respectively. We observed the expected bands at 25 kDa for CsgD and 27 kDa for OmpR234. Cultures carrying BBa_K2229300 (CsgD and OmpR234 expression), however, showed two extra bands at 15 kDa and 30 kDa, which were not observed in cultures expressing either CsgD or OmpR234 alone. We looked into the other curli operon genes, and found that CsgG is around 30 kDa, whereas CsgA, B, C, E, and F are all around 15 kDa (Robinson et al. 2006; Uhlich et al. 2009; Shu et al. 2012). This suggests that, as expected, BBa_K2229300 stimulates the production of other curli proteins as well (predicted proteins and sizes are labeled in the figure below).

CONGO RED ASSAY

After confirming protein expression, we wanted to test if our constructs actually lead to faster and more robust biofilm production. 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. The samples were then washed with PBS and dried. Any stained biofilm on the glass coverslips was solubilized in ethanol, and absorbance was measured at 500 nm. If biofilms were present, the solution would appear red, which could be quantified by an absorbance value. When all three expression constructs were compared, we find that overexpression of OmpR234 and CsgD together (BBa_K2229300) increased biofilm production the most. BBa_K2229300 also increased adhesion to glass coverslips, and we could see a layer of biofilm which remained attached to the glass surface after the washing steps.

Overexpression of both CsgD and OmpR234 (BBa_K2229300) increases biofilm production the most, compared to bacteria only expressing one of the proteins. A) Congo red assay stains biofilms. BBa_K2229300 increases adhesion to glass surfaces. B) Stained biofilm is solubilized in ethanol. C) Absorbance is measured at 500 nm.

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 877
  
• 1000
  COMPATIBLE WITH RFC[1000]