Team:TAS Taipei/Improve
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Project
Experiments
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Prototype
Human Practices
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About Us
Attributions
Project
Experiment
Modeling
Prototype
Human Practice
Safety
About Us
Attributions
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IMPROVE AN EXISTING PART
Our new composite part BBa_K2229300 improves the function of two existing parts: BBa_K342003 (ompR234 ORF) and BBa_K805015 (csgD ORF). CsgD and OmpR234 are regulators of two curli operons, which contribute to biofilm formation. When both proteins are overexpressed, we hypothesized that twice the amount of curli monomers should be made and exported to form fibers and biofilm. When we compared protein expression using SDS-PAGE, we found that BBa_K2229300 stimulated the expression of more curli proteins compared to samples that only expressed CsgD or OmpR234 alone. Using Congo Red, a dye commonly used to measure biofilm production, we also found that overexpression of both OmpR234 and CsgD (BBa_K2229300) increased biofilm production and adhesion to glass coverslips the most. These results show that the combination of BBa_K342003 and BBa_K805015 in a new composite part improves the individual functions, and BBa_K2229300 increases the yield of biofilm production.
BBa_K2229300
Figure 3-12 CsgD and OmpR234 Expression Our construct includes a strong promoter, two strong RBS, csgD, ompR234 and double terminator. Figure: Justin Y.
The existing parts BBa_K342003 (ompR234 ORF) and BBa_K805015 (csgD ORF) were taken from the distribution kit. First BBa_K805015 was inserted behind a strong promoter + strong RBS (BBa_K880005) (figure 3-10) to make the intermediate BBa_S05397. At the same time, ompR234 was inserted before BBa_B0015 (figure 3-10) to make the intermediate BBa_S05398.
Figure 3-11. PCR Check for BBa_K880005+CsgD and OmpR234+BBa_B0015. The expected size of BBa_K880005+CsgD is 1000 bp (orange box) and OmpR234+BBa_B0015 is 1100 bp (blue box). Cloning: Catherine Y., Dylan L., Justin Y.
Figure 3-13 PCR Check for BBa_K2229300. The expected size of BBa_K2229300 is 1900 bp (green box) Cloning: Catherine Y., Dylan L., Justin Y.
To make the final composite, a strong RBS (BBa_B0034) was inserted in front of BBa_S05398 to make BBa_S05399. FInally, BBa_S05397 was inserted before BBa_S05399 to complete the full construct BBa_K2229300 (figure 3-13). Sequencing results from Tri-I Biotech confirmed that our final construct is correct.
HYPOTHESIS
Figure 3-14 Overexpression of CsgD and/or OmpR234 upregulates the curli operon to different degrees We hypothesized that biofilm production would be upregulated (in increasing order) if we overexpress A) CsgD, B) OmpR234, or C) both. Figure: Justin Y.
We hypothesized that biofilm production would be upregulated (in increasing order) if we overexpress CsgD, OmpR234, or both (figure 3-14). Overexpression of CsgD would result in more curli monomers, but no transport proteins to carry the monomers out of the cell. Overexpression of OmpR234 would allow curli monomers to be exported and form curli fibers and biofilm. Finally, when both CsgD and OmpR234 are overexpressed, twice the amount of curli monomers should be made and exported to form even more curli fibers and biofilm.
SDS-PAGE Gel
The original parts, BBa_K342003 (ompR234 ORF) and BBa_K805015 (csgD ORF), were cloned into expression devices, Bba_K2229200 and Bba_K2229100, respectively. We observed the expected bands at 25 kDa for CsgD and 27 kDa for OmpR234. Cultures carrying BBa_K2229300 (CsgD and OmpR234 expression), however, showed two extra bands at 15 kDa and 30 kDa, which were not observed in cultures expressing either CsgD or OmpR234 alone. 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 other curli proteins as well (predicted proteins and sizes are labeled in figure 3-15).
Figure 3-15. SDS-PAGE results show that BBa_K2229300 overexpress both CsgD and OmpR234, as well as other proteins from the curli operons. Predicted proteins and sizes are listed on the right. E. coli expressing GFP was used as a positive control. Protein Gel & Figure: Justin Y.
Congo Red Assay
After confirming protein expression, we wanted to test if our constructs 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 transferred to 12-well plates with glass coverslips and incubated at 37˚C for one day. The samples were then washed with Phosphate Buffered Saline (PBS) and dried. Any stained biofilm on the glass coverslips was solubilized in ethanol, and absorbance was measured at 500 nm (figures 3-19). If biofilms were present, the solution would appear red, which could be quantified by an absorbance value.
Overexpressing CsgD or OmpR234 increased biofilm production, compared to controls with only csgD or ompR234 ORFs, as we hypothesized (figures 3-16 and 3-17). When all three expression constructs were compared, we find that overexpression of OmpR234 and CsgD together (BBa_K2229300) increased biofilm production the most (figure 3-19). BBa_K2229300 also increased adhesion to glass coverslips, and we could see a layer of biofilm which remained attached to the glass surface after the washing steps (figure 3-19, A).
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.
