CHARACTERIZATION OF BBa_K342003 & BBa_K805015

In order to characterize these existing parts, we cloned them behind a promoter, RBS, and in front of a transcriptional terminator (figures 3-8 and 3-9). PCR checks showed that cloning was successful (figure 3-11). Sequencing results from Tri-I Biotech confirmed that both of our constructs are correct.

SDS-PAGE Gel

SDS-PAGE was performed using transformed and lysed E. coli cultures. Bacteria overexpressing CsgD and OmpR234 showed darker bands at 25 kDa and 27 kDa, respectively, compared to controls that only contain the respective ORFs.

Congo Red Assay

To test if the overexpression of CsgD or OmpR234 actually lead to faster and greater 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 grown in 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 and 3-17). If biofilms were present, the solution would appear red, which could be quantified by an absorbance value.

We find that overexpressing either CsgD or OmpR234 increases biofilm production, as we hypothesized (figure 3-16 and 3-17). In our experiments, overexpression of CsgD leads to about twice the amount of biofilm compared to BBa_K850015 (figure 3-16), whereas overexpression of OmpR234 leads to about 8 times more biofilm compared to BBa_K342003 (figure 3-17). When bacteria expressing OmpR234 was grown in petri dishes, biofilms also appeared thicker compared to controls (figure 3-18).