Part:BBa_K1092025:Design
T7-RBS -ssDsbA-PDZ Ligand-Voltage Switch-Laccase
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 929
Illegal AgeI site found at 2447 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 550
Illegal BsaI.rc site found at 2608
Design Notes
Controlling rate of degradation of PAH with the voltage switch:
Rate of enzymatic reactions are limited by the diffusion rate of substrate. In normal experimental setting or physiological conditions, this rule hold true, and that’s why enzymatic activities are usually not very high, because all the enzymes and substrates are circulating in the medium in a chaotic manner. In our project, we demonstrate a way to alter the substrate diffusion rate by controlling the diffusion distance through electricity, thereby controlling the rate of reactions. We picked Laccase and Dioxygenase as an example because they are a pair of enzymes that can degrade carcinogenic substances: Polycyclic Aromatic Hydrocarbons (PAH) into simple and carboxylic acids that are either non-toxic or only slightly toxic. When the switch is OFF, the enzymes are too far away, and so the reaction rate is limited by the diffusion rate of the substrate, which mean it is slow. When the switch turns ON, the two enzymes are brought together, which greatly reduce the diffusion distance of substrate, and thereby increase the reaction rate.
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.