Team:Toronto/Results
Results
Successful Results
- 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 specific to gold
- Long GolS (BBa_K2048001) is more responsive to both gold and copper concentrations than York 2013 construct (BBa_K1127008)
- Long GolS (BBa_K2048001) was found to be significantly more response to gold than it is to copper
Unsuccessful Results
- Cell-free assay required further modifications
Future plans and direction
- Make Long GolS P118A (BBa_K2048002) and Long GolS (BBa_K2048001) work in a cell-free environment
- Integrate that cell-free environment onto a paper-based system (first fresh paper-based system and then freeze-dried paper-based system)
Discussion
The iGEM Toronto 2016 team was successful at modifying an naturally existing transcription factor called 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). For further details on what is contained in the Long GolS P118A BioBrick, see Figure 1.
To characterize our plasmids, we compared the expression levels of our Long GolS P118A (BBa_K2048002) and Long GolS (BBa_K2048001) against York 2013's GolS (BBa_K1127008) and a DH10\beta with varying concentrations of gold and copper.
Figure 2 shows the 96-well plate that was used in our BioBrick characterization. First comparing our constructs to York 2013's construct, our GolS (BBa_K2048001) was able to more strongly express LacZ in both presence of gold and copper compared to York 2013's GolS (BBa_K1127008). This can be determined by how wells G1 through G5 are more red in comparison to wells E1 through E5 for gold and C1 through C5 compared against A1 through C5 for copper. This can also be visualized in Figure 3 where the assay was at 24 hours.
Secondly, when looking at the assay, the degree of how red a well is becomes weaker or more yellow after 0.5\muM. 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 (Figure 4). 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 5. We speculated that this effect might be minimum for cell free extracts that contain minimal enzymes.
Thirdly, we compared our GolS (BBa_K2048001) and GolS P118A (BBa_K2048002) constructs response to and in the presence of gold and copper. Based on observations of 12 hours (Figure 2) and 24 hours (Figure 3), it is visible that the GolS (BBa_K2048001) from G1 through G5 (the Au(3) concentrations) is darker red compared to C1 through C5 (the Cu(2) concentrations). The visible significant difference in the darker shades of red for the gold concentration wells compared to copper indicates that our constructs are far more sensitive to gold than it is to copper. Figure 6 shows the GolS (BBa_K2048001) construct change in expression in the presence and response to gold over time.
Finally, for the GolS P118A (BBa_K2048002) expression in response to and presence of gold compared to copper, both the assay after 12 hours (Figure 2) and 24 hours (Figure 3) show that the GolS P118A (BBa_K2048002) is specific to gold with minimal to no expression in the presence of copper. The visible red in the copper wells are minimal indicating that it appears our amino acid change was successful at significantly decreasing the sensitivity of the genetic circuit expression in the presence of copper. Figure 7 shows the GolS P118A (BBa_K2048002) construct change in expression in the presence and response to gold over time.
Unfortunately, our cell-free assay did not work and we would have required more time to troubleshoot our protocol to determine why it did not success. A possible reason for our lack in success in our cell free assays was that we found that the NEB PureExpress (a cell-free transcription and translation system for E. coli translation) may have required modifications towards plasmids/BioBricks (such as possibly including a T7 promoter). Please refer to the notebook section for what we attempted to do.
A future direction this research may encourage is to take our GolS P118A (BBa_K2048002) BioBrick and be able to create a cell-free paper-based system as it would allow an alternative, cheap and quick method for the detection of gold. Our BioBrick has already been modified (Gols P118A, BBa_K2048002) and characterized to have a decreased sensitivity to copper compared to its unmodified version (GolS, BBa_K2048001).
