Team:NUS Singapore/Demonstrate
Demonstrate
1) We had successfully constructed the temperature-sensitive system with GFP reporter (BBa_K2447014). The construct was functioning properly and elucidating higher GFP expression only when the temperature was higher than 36 °C. ✔
2) Modelling has helped us to decide which designs to proceed with construction. ✔
3) Through modelling insights, we had successfully constructed the improved version of the extracellular phosphate sensor with GFP reporter (BBa_K2447000). The construct was functioning properly and elucidating higher GFP expression only when the phosphate concentration is minimal. ✔
4) We had successfully constructed the blue light activated inducible system with RFP reporter (BBa_K2447501). The construct was functioning properly and elucidating higher RFP expression only when blue light was administered. ✔
5) Over the course of the experimentation, although we did not manage to characterise the two-inputs (phosphate-temperature) cascaded system with controlled IM2 expression construct (BBa_K2447016), our team had successfully constructed and characterised the phosphate-temperature cascaded system with GFP reporter (BBa_K2447015)], to mimic the controlled production of IM2 proteins (immunity protein for E2 killing protein) under different temperature and phosphate conditions. ✔
Assuming E2 killing protein is constitutively produced and interacts in a 1:1 ratio with IM2 protein, higher GFP production would correlate with higher production of IM2 protein and thus enabling the cells to survive when the kill switch was in the ‘OFF’ states. Preliminary results had shown that phosphate-temperature system was partially working and yielding much higher GFP productions in 2 of the 3 non-killing conditions (Figure 1) and yielding 50% lower GFP productions in the killing condition of low phosphate and low temperature. However, in one of the non-killing condition (Figure 1: high phosphate and low temperature) where GFP production was predicted to be high, the actual GFP production was much lower and similar to the GFP production in the killing condition (low phosphate and low temperature). From the experimental results , we hypothesized that one of the reasons GFP production was lower than expected was because the phosphate promoter was leaky in its repressive states, and produced the temperature sensitive Tlpa36 proteins even in the non-killing states; leading to unwanted repression of the downstream temperature sensitive promoter. For future works, for a start, the phosphate promoter binding strength could be fine-tuned by means of directed mutagenesis to screen for tighter versions of the promoter.
6) We had successfully demonstrated that our proposed standardized methodology for designing kill switches was feasible. Through our modelling framework, it had proactively guided the experimental design. ✔
7) We had successfully constructed the plasmid that expressed IM2 protein (anti-toxin) under the control of our phosphate-temperature sensor (BBa_K2447016). But due to time constraint, we did not proceed with the experimentation with E2 protein (toxin) expression (BBa_K2447017) and thereafter, experimentation to study the E2-IM2 interactions in eventual double plasmids kill switch system. ✔
Figure 1: Bba_K2447015 Phosphate-temperature sensitive cascaded system with GFP reporter, experimentation with a microplate reader for continuous 8 hours incubations at both 37 °C and 30 °C. The amount of GFP production is supposed to predict the amount of IM2 proteins (immunity protein for E2 killing protein) that would be produced in Bba_K2447016. Under the three non-killing conditions (at high phosphate concentrations or at low phosphate concentrations but high temperature), GFP productions were expected to be high and similar in values. These would reflect the high amount of IM2 proteins produced to offset the constitutive production of the E2 killing protein, allowing the cells to stay alive. However, as seen in the high phosphate and low-temperature condition, the system is responding to the temperature stimulus but not responding to the phosphate one. Optimization works would have to be performed to balance the promoter, in order to achieve the dual input response. Under the killing condition (low phosphate concentration and low temperature), GFP production was low as expected, suggestive of the lower level of IM2 production in the kill switch construct, enabling the E2 killing protein to accumulate and kill the cells.
