Difference between revisions of "Part:BBa K2229100"

Revision as of 04:55, 13 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.

Characterization

Expression of CsgD Increases Biofilm Formation

To test the expression of CsgD, we ran SDS-PAGE using transformed and lysed E. coli cultures (figure 3-15). A culture transformed with the basic part BBa_K805015 (csgD ORF alone) was used as a negative control. We expected to see CsgD around 25 kDa (Brombacher et al. 2006; Martinez & Stock 1997). Compared to negative control, thicker and darker bands at the expected sizes were observed with both BBa_K2229100 (CsgD overexpression) (figure 3-15; proteins bands are marked by asterisks). In addition to the bands at 25 and 27 kDa, cultures carrying BBa_K2229300 (CsgD and OmpR234 expression) contained two extra bands at 15 kDa and 30 kDa, which were not observed in the negative controls. 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 figure 3-15).

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 3-16, 3-19). If biofilms were present, the solution would appear red, which could be quantified by an absorbance value. We find that overexpressing CsgD increases biofilm production, as we hypothesized (figure 3-19). Overexpression of CsgD (BBa_K2229100) doubles biofilm production compared to the negative control BBa_K805015 (figure 3-16).

Fig. 3-16

Fig. 3-19

When three constructs (BBa_K2229100, BBa_K2229200, BBa_K2229300) were compared, we find that overexpression of CsgD alone upregulated biofilm production, but not to the same degree as BBa_K2229200 and BBa_K2229300.

Sequence and Features