Revision as of 07:25, 30 November 2017

CsgD Expressing Construct

A CsgD-based construct that employs the strong promoter/strong RBS (K880005) combination to upregulate expression of CsgD, a transcriptional regulator that activates the synthesis of curli fibers and biofilm formation in Escherichia coli.

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 using the forward and reverse primers VF2 and VR. The expected PCR 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.

References<h1> Reinke, A A, and J E Gestwicki. “Insight into amyloid structure using chemical probes.” Chemical biology & drug design., U.S. National Library of Medicine, June 2011, www.ncbi.nlm.nih.gov/pubmed/21457473. Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]