Revision as of 07:54, 28 October 2017

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. 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 end transcription.

For strong CsgD expression (BBa_K2229100), csgD was inserted behind BBa_K880005 (BBa_S05397), and then before BBa_B0015.

PCR Check Gel

PCR Check for BBa_K2229100. The expected size of BBa_K2229100 (CsgD full construct) is 1100 bp (orange box).

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