Part:BBa_K4256666

BluetL (Blue light & Low Temperature) Bistable-Switch

Description

Figure 1a. Figure 1. The 2×2 transfer switch. mCherry and YFP are embedded as cargo and reporters.

The UCAS-China iGEM team 2022 designed a bistable switch controlled by both blue light and temperature. The switch combines a photosensitive, blue light-controlled switch [https://parts.igem.org/Part:BBa_K2469004 (BBa_K2469004)]with two RBSs corresponding to Temperature（25°C） [https://parts.igem.org/Part:BBa_K3482001 (BBa_K3482001)]binding at both sides of the switch. When the temperature rises to 25 ℃ or higher, the RC site (RNase E cleavage site) of the RBS will be truncated by RNase E, which will stop the expression of the downstream gene. At low temperatures(≤25°C), the ARC site (Anti RNase E cleavage site) matches with the RC site complementarily and forms a stem ring structure, which can not be cut by ribonuclease. Thus, the target gene downstream of the Ptrc promoter can express. This construct demonstrates a new configuration of a 2*2 bistable switch. Sequence and Features BBa_K4256666 SequenceAndFeatures

Design & Experiment

To verify the feasibility of the switch, The UCAS-China iGEM team 2022 designed two plasmids (pET-28a) containing our switch sequences. With four different fluorescent proteins added as reporters, we can quantify the feasibility of our design. In the absence of blue light, YFP (yellow) and EYFP (cyan) proteins will be produced at 37°C and 25°C respectively. In contrast upon blue light excitation, the reporter located at the other side of the switch will separately fabricate mCherry (red) and JGFP (green) proteins at 37°C and 25°C. We managed to transform our plasmids into the competent cell (DH5) and successfully observed the expression of the report proteins through the fluorescence microscope (Results are as followed).

Figure 1a. the result of the bistable-switch.

Due to the coronavirus pandemic and inadequate staffing, we only validate the practicability of each pathway in our switch. The changes between different statuses of our switch have not been completely texted. Within our design, the switch should be changed along with the existence of the blue light.

Future experiment & redesign

When choosing our reporters, we focused on fluorescent proteins (For example EYFP) instead of Chromoproteins, which caused a lot of difficulty in utilizing the fluorescence microscope to obtain results. Additionally, we did not consider the quenching of fluorescent proteins and neglected the damage to the cells done by these proteins. We plan to change our reporter proteins with Chromoproteins in our future design. For more information, please visit: UCAS-china/proof-of-concept.