Team:CU-Boulder

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Project Overview

Improving Ethanolamine Utilization Compartments

Bacterial microcompartments (BMCs) 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 ethanolamine utilization compartment (EUT), by introducing a non-natural amino acid into the outer shell protein. A previous iGEM gene has already taken advantage of this compartment, but by minimizing the necessary proteins we have found that it is still possible to form a EUT compartment by expression of only the EutS portion of the gene. This 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. The formation of compartments will be visualized through EutC tagged eGFP localization within the Euts compartments. Various levels of EutCeGFP and EutS expression have led us to an optimal combination that allows the formation of at least one compartment with enough fluorescence to see, but not so much that its bleaches the resulting image.

Fig. I: (A). EUT-operon diagram (asterisks indicate genes with proposed N-termini targeting peptide sequences—EutG N-terminal sequence was not shown to target assembling EUTs); (B). EUT BMC functional schematic indicating the putative biochemical pathway for the catabolism of cytotoxic ethanolamine substrate into biologically-inert products including ethyl alcohol, acetyl-phosphate, and acetyl-CoA. Figure courtesy Choudhary et alii research team; cited from pp. 3 of Engineered Protein Nano-Compartments for Targeted Enzyme Localization in PlosOne.

Controlling Ethanolamine Utilization Compartments

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-phenylalanine, a structurally differing but functionally equivalent amino acid, 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, as shown below, but the laser used to excite eGFP may also cause conformational change of azo- phenylalanine. 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.