Team:CU-Boulder

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Project Description

Bacterial microcompartments (BMC’s) occur in nature to encapsulate enzymatic and metabolic processes in organisms such as E.coli. This capsulizing system can also be utilized to efficiently deliver drugs to targeted regions. Our goal is to engineer the reversible assembly and disassembly of an ethanolimine utilization compartment (EUT), by introducing a non-natural amino acid into the outer shell protein. The EutS gene codes for a protein that forms the hexameric tiles which make up the EUT compartment shell and can associate with EutC tagged proteins. Azo-benzene, the non-natural amino acid we will be using, undergoes a conformational change when exposed to specific wavelengths of light and will be used to disrupt the EutS structure. The formation of compartments will be visualized through EutC tagged eGFP localization within the Euts compartments and the destruction of the compartments will be indicated by EutC-eGFP dispersion throughout the cell.

Previous research has shown successful changes in chaperonin conformation by crosslinking cysteine residues with a light activated azobenzene - dimaleimide. At 450 nm azo-benzene will primarily be in its longer trans state but when exposed to 365 nm light, azo-benzene has a pinched cis conformation. We will be replacing amino acids within the EutS structure with azo-benzene to create steric hindrance caused by cis to trans isomerization rather than crosslinking. If placed correctly in the EutS protein azo-benzene may control the formation of the BMCs.

Successful microscopy has confirmed the viability of EutS and EutC-eGFP in E.Coli, but the laser used to excite eGFP may also cause conformational change of azo-benzene. Instead Neptune, another light activated protein that is excited at a much longer wavelength, will be used to visualize the formation and destruction of EutS microcompartments. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance.

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Successful microscopy has confirmed the viability of EutS and EutC-eGFP in E.Coli, but the laser used to excite eGFP may also cause conformational change of azo-benzene. Instead Neptune, another light activated protein that is excited at a much longer wavelength, will be used to visualize the formation and destruction of EutS microcompartments. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance. Instead Neptune, another light activated protein that is excited at a much longer wavelength, will be used to visualize the formation and destruction of EutS microcompartments. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance.Instead Neptune, another light activated protein that is excited at a much longer wavelength, will be used to visualize the formation and destruction of EutS microcompartments. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance. Future work will focus on the implementation of a multiconstruct system with EutS, EuC-Neptune, and a third construct that creates tRNA’s to integrate azo-benzene into the EutS at locations we expect to see significant steric hinderance.

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