Team:Toronto/Results
Results
Success
- Both Long GolS P118A (BBa_K2048002) and Long GolS (BBa_K2048001) constructs responded to gold concentrations in a dose-responsive manner up to 1uM
Long GolS P118A (BBa_K2048002) is more responsive to gold than copper
Figure 1: CPRG assay results after 12 hours of incubation Long GolS (BBa_K2048001) is more responsive to both gold and copper concentrations than York 2013 construct (BBa_K1127008)
Discussion
We expected to see a linear increase correlated to increase in the concentration. However, instead, we observed a sudden decrease in expression levels after 0.5uM. This became more pronounced over time, resulting a Range Detector Circuit.
We suspect that there is an inherent edge that represses the expression after a certain threshold. This would produce the circuit shown in Figure 4. We speculated that this effect might be minimum for cell free extracts that contain minimal enzymes.
Not so successful
- Cell-free assay required more time and troubleshooting.
Future plans
- Make cell-free assays work.
The iGEM Toronto 2016 team was successful at modifying an naturally existing transcription factor, GolS (natively found in Salmonella enterica), to contain certain mutations that have been found to cause CueR (a copper-response factor in Escherichia coli)to increase sensitivity to gold and significantly decrease it's sensitivity to copper. This modification was due to an amino acid change from proline to alanine at site 118. This successful synthetic change can be found composite BioBricks, Long GolS P118A (BBa_K2048002) compared to the unchanged Long GolS (BBa_K2048001).
