Part:BBa_K805015
CsgD, the master regulator of biofilm formation
CsgD, the master regulator of biofilm formation, activates the synthesis of curli fimbriae and extracellular polysaccharides in Escherichia coli.
Characterization
Team: TAS_Taipei 2017
Author: Justin Yang
SDS-PAGE
BBa_K2229100 contains and expresses CsgD (BBa_K342003). 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_K342003. 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.
BBa_K2229300 Improves the Function of BBa_K805015
SDS-PAGE
Our new composite part BBa_K2229300 improves the function of two existing parts: BBa_K342003 (ompR234 ORF) and BBa_K805015 (csgD ORF). CsgD and OmpR234 are regulators of two curli operons, which contribute to biofilm formation. When both proteins are overexpressed, we hypothesized that twice the amount of curli monomers should be made and exported to form fibers and biofilm.
CONGO RED ASSAY
Cultures carrying BBa_K2229300 (CsgD and OmpR234 expression) 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 all curli proteins (predicted proteins and sizes are labeled in the SDS-PAGE figure above).
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |