Characterization of our prototype

Figure. The characterisation result of the prototype of the tristable switch. This result was obtained by combining 3 characterization data obtained in 3 different days. Error bars represent SEM from 3 biological replicates.

The results showed that the Tristable switch could neither produce steady output nor possess sensitive state-changing ability as we expected. Three different inducers, aTc, DAPG and IPTG were added to induce one of the three promoters every two hours in order to test its capacity to switch after induction. Unfortunately, the construct did not exhibit these characteristics while green fluorescence proteins keeping on dominating throughout the whole experiment. According to the data obtained, both the levels of red fluorescence protein and blue fluorescence protein were significantly lower than that of green fluorescence protein without responding to its specific inducers.

We therefore conjectured that this might be due to the imbalance promoter strength among the three promoters and the insufficient induction time. Mutations could be excluded since sequencing of the circuit was done and proved to be correct.

1. Three different repressible promoters, tetp, phlFp and lacpwere used to build the circuit. lacp were the strongest one among the three according to the paper, Genomic mining of prokaryotic repressors for orthogonal logic gates. Since GFP was driven by lacp and the GFP level remained nearly the same throughout the whole experiment, it suggested that the promoter strength of lacp was too strong that the other two promoters were continuously inhibited by the repressors that were driven by the lacp.

2. The induction time after adding inducers might not be enough. If time was not enough, it would be hard to produce the fluorescence proteins to a particular level. Usually, the induction time would take around 6 hours which is triple compared to ours (Cameron, 2014). If there was not enough time for the circuit to produce sufficient amount of fluorescence proteins, it would be hard to measure the fluorescence accurately. Aside from this, some repressor proteins would remain, thus, hamper the activation of another repressible promoter.

Further Improvments

1. In order to build a circuit that could produce steady output, strength of most of the parts involved should be kept constant, if not all. In our case, the ribosome binding sites in the construct could be replaced by a stronger or weaker one depending on the situation. This fine tuning process would help regulate the overall strength to avoid any part from dominating.

2. The induction time would be extended. In this case, we could assume all the repressors produced in the last induction has degraded while enough time is provided to produce sufficient fluorescence proteins for the assay.

Reference

1. Cameron, D. E., &a Collins, J. J. (2014). Tunable protein degradation in bacteria. Nature biotechnology, 32(12), 1276-1281.
2. Stanton, B. C., Nielsen, A. A., Tamsir, A., Clancy, K., Peterson, T., & Voigt, C. A. (2014). Genomic mining of prokaryotic repressors for orthogonal logic gates. Nature chemical biology, 10(2), 99-105.