Part:BBa_K2229100
CsgD Expressing Construct
A CsgD-based construct that employs the strong promoter/strong RBS (K880005) combination to up-regulate expression of CsgD, a transcriptional regulator that activates the synthesis of adhesive curli fimbriae in Escherichia coli, which up-regulates biofilm formation.
Construct Design
This construct was built to upregulate curli production by overexpressing CsgD (BBa_805015). 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), and a double terminator (BBa_B0015) to
Characterization
SDS-PAGE
BBa_K2229100 contains and expresses CsgD (BBa_K805015). SDS-PAGE results show CsgD protein around 25 kDa, which matches the expected size.
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 CsgD (BBa_K2229100) in our experiments doubles biofilm production compared to the control BBa_K805015. When bacteria expressing CsgD were plated in petri dishes, biofilms appeared thicker compared to controls.
Overexpression of CsgD (BBa_K2229100) doubles biofilm production. A) Congo red assay stains biofilm (red). B) Stained biofilm is solubilized in ethanol. C) Absorbance is measured at 500 nm.
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]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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