Difference between revisions of "Team:CU-Boulder"

 
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<h2> Put it in a Box</h2>
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<h3> Project Overview </h3>
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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.</p>
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Bacterial microcompartments (BMCs) occur in nature to encapsulate enzymatic and metabolic processes in organisms such as <i>E.Coli</i>. We have focused our work on the simplest BMC, the ethanolamine utilization compartment (EUT). Our goal is to engineer the an improved and <a href="#999">simplified EUT</a> that requires minimal genes and can <a href="#9999"> assemble and disassemble</a> by the introducing a non-natural amino acid into the outer shell protein.</p>
  
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<p class="main"> 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. </p>
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<p class = "main"> 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- 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. </p>
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<h2> Improving Ethanolamine Utilization Compartments </h2>
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Previous iGEM teams have already taken advantage of the compartment formed by the EutSMNLK operon, but we have found that it is still possible to form a EUT compartment by expression of only the EutS gene. This EutS gene codes for a protein that forms the hexameric tiles which make up the EUT compartment shell and can associate with and encapsulate EutC1-19, a component of the EutC gene, and any proteins attached to it. The formation of these compartments will be visualized through this EutC tagged eGFP localization within the EutS compartments. Various levels of EutC-eGFP and EutS expression have led us to an optimal combination that allows the formation of at least one compartment per <i>E.Coli</i> cell with optimal fluorescence for visualization. </p>
  
 
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<figcaption align="center"> EutS eGFP Compartmentalization </figcaption>
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<figcaption float="center" style = "margin-left: 600px; width: 48em;">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 <i> Engineered Protein Nano-Compartments for Targeted Enzyme Localization </i> in PlosOne. </figcaption>-->
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<h2> Controlling Ethanolamine Utilization Compartments </h2>
 +
<p class="main"> <a name="9999"></a>
 +
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. This previous research demonstrated that taking advantage of these two conformations can be used to "close" and "open" a chaperonin. Towards the same end, we will be replacing amino acids within the EutS structure with azo-phenylalanine, a similar 'azobenzene' chemical structure linked to a phenylalanine amino acid, to create steric hindrance caused by cis to trans isomerization. If placed correctly in the EutS protein, this azobenzene containing residue could be used to prevent or allow the formation of EutS compartments. </p>
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<p class = "main"> Successful microscopy has confirmed the viability of EutS and EutC-eGFP in <i>E.Coli</i>, as shown below, but the laser used to excite eGFP may also cause conformational change of azo-phenylalanine. In the future we hope to use a fluorescent protein in the far-red spectrum to visualize the formation and destruction of EutS microcompartments. Our future work will focus on the implementation of a multiconstruct system with EutS, EutC1-19 with a new far-red fluorescent protein, and a third construct that creates tRNA’s to integrate azobenzene into the EutS at locations we expect to see just the right level of steric hindrance. </p>
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<figcaption style = "margin-left: 70px;padding: 10px 10px 10px;">Fig.1 - Jonah and his creepy smile</figcaption>
 
<figcaption style = "margin-left: 70px;padding: 10px 10px 10px;">Fig.1 - Jonah and his creepy smile</figcaption>
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Latest revision as of 23:40, 19 October 2016

Put it in a Box

Bacterial microcompartments (BMCs) occur in nature to encapsulate enzymatic and metabolic processes in organisms such as E.Coli. We have focused our work on the simplest BMC, the ethanolamine utilization compartment (EUT). Our goal is to engineer the an improved and simplified EUT that requires minimal genes and can assemble and disassemble by the introducing a non-natural amino acid into the outer shell protein.

Improving Ethanolamine Utilization Compartments

Previous iGEM teams have already taken advantage of the compartment formed by the EutSMNLK operon, but we have found that it is still possible to form a EUT compartment by expression of only the EutS gene. This EutS gene codes for a protein that forms the hexameric tiles which make up the EUT compartment shell and can associate with and encapsulate EutC1-19, a component of the EutC gene, and any proteins attached to it. The formation of these compartments will be visualized through this EutC tagged eGFP localization within the EutS compartments. Various levels of EutC-eGFP and EutS expression have led us to an optimal combination that allows the formation of at least one compartment per E.Coli cell with optimal fluorescence for visualization.

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. This previous research demonstrated that taking advantage of these two conformations can be used to "close" and "open" a chaperonin. Towards the same end, we will be replacing amino acids within the EutS structure with azo-phenylalanine, a similar 'azobenzene' chemical structure linked to a phenylalanine amino acid, to create steric hindrance caused by cis to trans isomerization. If placed correctly in the EutS protein, this azobenzene containing residue could be used to prevent or allow the formation of EutS compartments.

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. In the future we hope to use a fluorescent protein in the far-red spectrum to visualize the formation and destruction of EutS microcompartments. Our future work will focus on the implementation of a multiconstruct system with EutS, EutC1-19 with a new far-red fluorescent protein, and a third construct that creates tRNA’s to integrate azobenzene into the EutS at locations we expect to see just the right level of steric hindrance.