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From 2013hs.igem.org

Proteorhodopsin: Light Driven Proton Pump

The first gene that we will express in our E. coli is the proteorhodopsin protein. Proteorhodopsin works as a light driven proton pump. When expressed in a cell the proteorhodopsin takes the place of the electron transport chain. Proteorhodopsin is a transmembrane protein that is bound around a retinal. Retinal is an unsaturated hydrocarbon chain that is attached to a lysine side chain of the protein (Lys 231). When exposed to light, retinal (which is a chromophore) absorbs the light. This causes the one of retinal’s double bonds to isomerize. Isomerization is the process of a molecule rearranging its atomic structure and changing into a different molecule with the same atoms. The double bond that is isomerized changes from a trans to cis state (tans-cis isomerization). Trans is when substituent groups are facing opposing directions, and cis is when they are facing the same direction. This isomerization in turn changes the retinal shape.

Retinal changes from a ground state to an excited state which causes surrounding changes in the protein. In the excited state of retinal, it releases a proton and it’s transferred into the aspartate side chain (Asp 97 which is the primary proton receiver). Asp 97 is located in the extracellular membrane of the cell and it hands off the proton via a path of water molecule transfers into the extracellular space. The now negatively charged retinal then takes in a proton received from another aspartate chain (Asp 227). Asp then replenishes its proton from the cytoplasm and the retinal returns to its ground state. The overall process can be simplified into one proton pumped, for every photo absorbed.

The proton pumping to the extracellular space of the cell then creates a proton gradient across the cell membrane. This causes chemiosmosis to occur, which is when there exists a gradient and protons from an area of higher proton concentration diffuse to an area of lower concentration. This transfer causes enough electrochemical potential in the ATP synthase to synthesize ATP molecules. Every three protons diffusing into the membrane result in one new ATP molecule synthesized. In the long run, the effect of the proteorhodopsin is harnessing sunlight in the form of photons and converting it into to ATP for energy.

The proteorhodopsin generator that we are designing involves four different parts. The promoter we are using is BBa_J23119 which is a strong promoter with evidence of success with proteorhodopsin. The RBS we are using is BBa_B0030, which is a strong RBS with successful experience. The proteorhodopsin gene we are using BBa_K572005 which was added to the parts registry by the Madras team from India in 2011. Our initial plan was to use BBa_B0012 and BBa_B0011 as the terminators but the parts registry classifies them with bad experience, so we made the shift to using BBa_B0014 which is a double terminator consisting of those part with better results. Our final generator would consist of all 4 parts and in a pSB1A3 plasmid backbone for ampicillin resistance.