Part:BBa_K2229300

Dual Expressing Construct of CsgD and OmpR234

The full construct that places a strong Promoter/RBS (BBa_K880005) in front of csgD (BBa_K805015), a transcriptional regulator of curli fimbriae and biofilm production. A Biobrick RBS (BBa_B0034) is inserted in front of the second protein OmpR234 (BBa_k342003), which promotes curli production by activating csgD. Both csgD and OmpR234 are acquired from the iGEM distribution kit, curli production is thus maximized through dual expression of the two proteins in Escherichia Coli. A double terminator (BBa_B0015) was added at the end to stop transcription.

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]

Characterization

Protein Gel

Cultures carrying BBa_K2229300 (CsgD and OmpR234 expression) contained two extra bands at 15 kDa and 30 kDa, which were not observed in the negative controls. 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 all curli proteins (predicted proteins and sizes are labeled in figure 3-15).

Fig. 3-15

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 (figures 3-19). If biofilms were present, the solution would appear red, which could be quantified by an absorbance value. We find that overexpressing CsgD and/or OmpR234 increases biofilm production to different degrees, as we hypothesized (figure 3-19). When all three constructs were compared, we find that overexpression of both OmpR234 and CsgD (BBa_K2229300) increased biofilm production the most (figure 3-19). BBa_K2229300 also increased adhesion to our glass coverslips, and we could see a layer of biofilm which remained attached to the glass surface after the washing steps (figure 3-19, A).

Fig. 3-19

In summary, we demonstrate that biofilms can trap NPs and our constructs function as hypothesized. Our collection of constructs (BBa_K2229100, BBa_K2229200, and BBa_K2229300) can successfully upregulate biofilm production to varying degrees.