Team:Hong Kong CUHK/vs

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 (Fig. 1). 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 (Fig. 2). 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.

Voltage Switch (Protein Domain BBa_K1092006)

It is a protein domain found in a transmembrane potassium ion channel from Aeropyrum pernix, which is highly voltage dependent. It contain positive charges from the 4 arginine residues, and also numerous leucine residues which give it high hydrophobicity. All these together make it an excellent voltage sensing peptide in the transmembrane region. Study has shown that this helical voltage sensor peptide is capable of moving across the membrane under external voltage, which is the key to the voltage dependence of the ion channel (PMID: 12721618). We make use of this feature to construct a novel voltage switch (BBa_K1092007 & BBa_K1092008), which consist of the PDZ domain (BBa_K1092102), PDZ ligand (BBa_K1092101), this voltage sensor peptide itself. To further adapt this voltage sensor peptide into our project, we added in two (GGGGS)3 flexible linker which serve to link other protein domains, and a (PE)5 linker which serve as a rigid rod that project from the sensor peptide to elongate it and increase the separation distance.

Part-only sequencing result:
(                      (GGGGS)3 linker                         )(
GGTGGTGGTG GTTCTGGTGG TGGTGGTTCT GGTGGTGGTG GTTCTCTGGG
Voltage sensor peptide
TCTGTTTCGT CTGGTGCGCC TGCTGCGTTT TCTGCGTATT CTGCTGATTA
)(                                      (Pe)5 linker           )(
TTTGCCCGGA ACCGGAACCG GAACCAGAAC CAGAAGGTGG TGGTGGTTCT
(GGGGS)3 linker                           )
GGTGGTGGTG GTTCTGGTGG TGGTGGTTCT

Note: part name should be voltage sensor peptide, not voltage switch

ssDsbA-PDZ Ligand-Voltage Switch (Protein Domain BBa_K1092007)

It is a part of the voltage switch, which consist of the PDZ Ligand (BBa_K1092101) and the voltage sensor peptide (BBa_K1092006). PDZ Ligand can form dimers with PDZ domain (BBa_K1092102), which links this part together with the PDZ domain-Voltage sensor peptide (BBa_K1092008).

ssDsbA-PDZ Domain-Voltage Switch (Protein Domain BBa_K1092008)

It is a part of the voltage switch, which consist of the PDZ domain (BBa_K1092102) and the voltage sensor peptide (BBa_K1092006). PDZ Domain can form dimers with PDZ Ligand (BBa_K1092101), which links this part together with the PDZ ligand-Voltage sensor peptide (BBa_K1092007).

RFP N terminus (Protein Domain BBa_K1092105)

An RFP fragment adapted from the RFP part BBa_E1010, and its counterpart is the RFP C terminus (BBa_K1092106). It is adapted to be expressed downstream of a fusion protein such as our Voltage switch parts (BBa_K1092023 & BBa_K1092024). It is the N terminal of the RFP, and is capable of refolding with it’s counter part C terminal as demonstrated by the NYMU-Taipei 2010 iGEM team through the Biomolecular Fluorescent complementation (BiFC) to give fluorescent (BBa_K411103 & BBa_K411104). We adapt this protein to be the downstream of our voltage switch because we want it to become our effector of the switch.

RFP C terminus (Protein Domain BBa_K1092106)

An RFP fragment adapted from the RFP part BBa_E1010, and its counterpart is the RFP N terminus (BBa_K1092105). It is adapted to be expressed downstream of a fusion protein such as our Voltage switch parts (BBa_K1092023 & BBa_K1092024). It is the C terminal of the RFP, and is capable of refolding with it’s counter part N terminal as demonstrated by the NYMU-Taipei 2010 iGEM team through the Biomolecular Fluorescent complementation (BiFC) to give fluorescent (BBa_K411103 & BBa_K411104). We adapt this protein to be the downstream of our voltage switch because we want it to become our effector of the switch.