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 fimbriae 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 200
- 1000COMPATIBLE WITH RFC[1000]
Characterization
Expression of OmpR234 Increases Biofilm Formation
Protein Gel
To test the expression of OmpR234, we ran SDS-PAGE using transformed and lysed E. coli cultures (figure 3-15). A culture transformed with the basic part BBa_K342003 (ompR234 ORF alone) is used as a negative control. We expected to see OmpR234 around 27 kDa (Brombacher et al. 2006; Martinez & Stock 1997). Compared to the negative control, thicker and darker bands at the expected sizes were observed withBBa_K2229200 (OmpR234 overexpression) (figure 3-15; proteins bands are marked by asterisks).
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 3-17, 3-19). If biofilms were present, the solution would appear red, whi
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