Part:BBa_K1092024:Design
T7-RBS-ssDsbA-PDZ Domain-Voltage Switch-RFP N terminus
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 132
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 362
Design Notes
BiFC Switch:
This switch make use of the biomolecular fluorescent complementation as the basis. When the switch is OFF, the two RFP fragments are far away from each other. Since the two proteins express separately, the two RFP fragments would not interact with each other, and so there is almost no fluorescent signal. When we switch it ON, the two fragments would come together. When the fragments are close enough, they will refold to generate fluorescent. Up until now a major challenge of such system is that the mature fluorescent protein will become too stable to be separated, and so normally under mild condition, the BiFC is irreversible. In order to solve the problem, our system can be a solution. By exerting negative charge onto the environment, the separation of the two fragments will not be only depending on the mutual repulsion since the attraction to the negatively charged environment would dominate. This can hopefully provide enough extra tension to pull the two fragments apart.
The Voltage Switch:
The voltage switch is a novel protein switch that responds to external voltage. The switch itself consist of the PDZ Ligand-Voltage sensor peptide (BBa_K1092007) and the PDZ Domain-Voltage sensor peptide (BBa_K1092008), and can be linked to different effectors such as the Dioxygenase (BBa_K1092002) and Laccase (BBa_K1092004), or the RFP fragments (BBa_K1092105 & BBa_K1092106). Initially, the two proteins would express and localize onto the inner membrane of the bacteria. The two peptides would then come together forming a dimer. After that, due to the mutual repulsion of the positive charges in the two voltage sensor peptide, the two voltage sensor peptide would separate. This separate the two effectors down below the two peptide, causing either a long distance for the two effectors to interact, or a longer distance of diffusion of substrates. This represent the OFF stage of the voltage switch, and it could be enhanced by using negatively charged environment to further pull them apart.
During the ON stage, the environment become positively charged, and this environment exert a strong electrostatic repulsion on the two voltage sensor peptide so that they can be pushed together by overcoming the mutual repulsion between the peptides. This action brings the two downstream effectors together, which either provides short-enough distance for them to interact, or greatly reduces the distance of diffusion of the substrates, thus greatly enhance the reaction rate.