Team:TAS Taipei/Contribution
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Project
Experiment
Modeling
Prototype
Human Practice
Safety
About Us
Attributions
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IMPROVE CHARACTERIZATION OF EXISTING PART
We improved the characterization of BBa_K342003 and BBa_K805015, which contain the ompR234 ORF and csgD ORF, respectively. We chose to characterize these parts because we wanted to use biofilm to capture nanoparticles. We looked through the distribution kit, found these two parts which regulate biofilm production, and tested them. We characterize the protein sizes of OmpR234 (BBa_K342003) and CsgD (BBa_K805015), and show that expression of OmpR234 and CsgD both increase biofilm production.
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.
Figure 3-8 CsgD expression with a strong promoter + strong RBS combination. Figure: Justin Y.
Figure 3-9 OmpR234 expression with a strong promoter + strong RBS combination. Figure: Justin Y.
Figure 3-11. PCR check for CsgD and OmpR full constructs. The expected size of CsgD full construct is 1100 bp (orange box) and OmpR full construct is 1200 bp (blue box). Cloning: Catherine Y., Dylan L., Justin Y.
SDS-PAGE Gel
Figure 3-15 SDS-PAGE shows E. coli overexpressing CsgD or OmpR234. Predicted sizes of the curli proteins are listed on the right, and E. coli expressing GFP was used as a positive control. Protein Gel & Figure: Justin Y.
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).
Figure 3-16: 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. Experiment & Figure: Yvonne W.
Figure 3-17 Overexpression of OmpR234 (BBa_K2229200) leads to ~8 times more biofilm production than control (BBa. A) Congo red assay stains biofilm (red). B) Stained biofilm is solubilized in ethanol. C) Absorbance is measured at 500 nm. Experiment & Figure: Yvonne W.
