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..
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 877
- 1000COMPATIBLE WITH RFC[1000]
Characterization
SDS-PAGE
When CsgD or OmpR234 were overexpressed, we observed thick bands at 25 kDa and 27 kDa, respectively. Cultures carrying BBa_K2229300 (CsgD and OmpR234 expression) contained two extra bands at 15 kDa and 30 kDa, which were not observed in other lanes. 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 SDS-PAGE figure).
" width="600px
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.
| None |