Team:Yale/Parts

From 2014.igem.org

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<h1 style="margin-top:22px; font-size:50px;">Modeling</h1>
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<h1 style="margin-top:22px; font-size:50px;">Submitted Parts</h1>
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<tr><td colspan="4"><h2>Modeling E. coli growth producing a toxic compound</h2>
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We sought out to determine the optimal time to induce the E. coli in order to produce the largest quantity of antimicrobial peptides.  We hypothesized that the optimal induction time would be around mid-log, when the E. coli are growing fastest and there are enough bacteria to produce a significant amount of peptide before the population levels drop. To test this theory, we created a theoretical model using MATLAB, using E. coli logistical growth combined with exponential decay (due to the antimicrobial peptide) at different induction times, as represented in the graphic below.
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Our collection of submitted biobricks consists of:
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<ul style="list-style-type:square"> <li>Mussel foot protein (MFP) 1-5-1 sequence [combination of <i>Mytilus galloprovincialis</i> Foot Protein 5 (Mgfp-5) and <i>Mytilus Edulis</i> Foot Protein 1 (Mefp-1)]. <li>MFP with superfolder Green Fluorescence Protein (sfGFP).<li>MFP with our anti-microbial peptide, LL-37.<li> Entire construct of our anti-microbial adhesive peptide: 2XStrep_Flagtag--LL-37--Mussel Foot Protein--sfGFP. </ul>
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<i>Note all biobricks are in the pSB1C3 plasmid.</i>
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<tr><td colspan="2"><h2>Full Construct: <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_K1396000">BBa_K1396000</a></a></h2>
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The part is an coding sequence for an anti-microbial peptides linked to a mussel-foot protein-linked to superfolder GFP for localization. The mussel foot protein will anneal to surfaces as a wet glue and the antimicrobial domain is designed to interact with microbial membranes and interfere with membrane stability. In order to use this part you can produce it in a TAG recoded organism simultaneously expressing a Tyrosine supressor or L-DOPA orthogonal translational system. In order to purify you can use the 2X Strep tag and strep column and later cleave with enterokinase to remove the sequence supressing LL-37 antimicrobial action.</p>
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<td colspan="4" align="middle"><img src="https://static.igem.org/mediawiki/2014/thumb/d/d9/Presentation1model.jpg/800px-Presentation1model.jpg"></td></tr>
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<tr><td colspan="2"><h2>LL-37-MFP: <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_K1396001">BBa_K1396001</a></h2>
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The part is an coding sequence for an anti-microbial peptides linked to a mussel-foot protein. The mussel foot protein will anneal to surfaces as a wet glue and the antimicrobial domain is designed to interact with microbial membranes and interfere with membrane stability. In order to use this part you can produce it in a TAG recoded organism simultaneously expressing a Tyrosine suppressor or L-DOPA orthogonal translational system. In order to purify you can use the 2X Strep tag and strep column and later cleave with enterokinase to remove the sequence suppressing LL-37 antimicrobial action.
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This is an improvement on the Utah State biobrick <a href= "http://parts.igem.org/Part:BBa_K1162006">BBa_K1162006 </a> which consists of only the LL-37 peptide.</p>
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<tr><td colspan="2"><h2>Determining Optimal Time to Induce Expression</h2>
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<tr><td colspan="2"><h2>MFP-sfGFP: <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_K1396002">BBa_K1396002</a></h2>
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The part is an coding sequence for an anti-microbial peptides linked to a mussel-foot protein-linked to superfolder GFP for localization. The mussel foot protein will anneal to surfaces as a wet glue and superfolder GFP will allow for florescence imaging and localization. In order to use this part you can produce it in a TAG recoded organism simultaneously expressing a Tyrosine supressor or L-DOPA orthogonal translational system. In order to purify you can use the 2X Strep tag and strep column and later cleave with enterokinase to remove the sequence supressing LL-37 antimicrobial action.</p>
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<tr><td colspan="2"><h2>Mussel Foot Protein 1-5-1: <a href= "https://static.igem.org/mediawiki/2014/0/04/FP151.jpg">BBa_K1396003</a></h2>
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We simulated a 24 hour period and determined the optimal time to induce the cells is around mid-log (~7.5 hours). Inducing at this time maximizes production of the peptide.  The graph below shows E. coli growth with induction at different times.  They follow a logistic growth model until the inducer is added and then there is an exponential decay. </p>
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Recoded and codon optimized coding sequence for the mussel foot protein 151. TAG is recoded. In order to produce the protein co-express in cells contain either an L-DOPA orthogonal translation system or a Tyrosine suppressor.</p>
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<p>Overlayed with this graph is a plot of <strong>total production of of the peptide vs. time of induction</strong>, (with induction at every 6 minutes over a 24 hour period). The highest production of peptide over the lifespan of these bacteria is represented by the peak of this plot, which corresponds to induction at mid-log, as we previously hypothesized.<br/> The MATLAB code for our model can be found <strong>Here</strong>.
 
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Latest revision as of 03:39, 17 October 2014

Submitted Parts

Our collection of submitted biobricks consists of:

  • Mussel foot protein (MFP) 1-5-1 sequence [combination of Mytilus galloprovincialis Foot Protein 5 (Mgfp-5) and Mytilus Edulis Foot Protein 1 (Mefp-1)].
  • MFP with superfolder Green Fluorescence Protein (sfGFP).
  • MFP with our anti-microbial peptide, LL-37.
  • Entire construct of our anti-microbial adhesive peptide: 2XStrep_Flagtag--LL-37--Mussel Foot Protein--sfGFP.

Note all biobricks are in the pSB1C3 plasmid.

Full Construct: BBa_K1396000

The part is an coding sequence for an anti-microbial peptides linked to a mussel-foot protein-linked to superfolder GFP for localization. The mussel foot protein will anneal to surfaces as a wet glue and the antimicrobial domain is designed to interact with microbial membranes and interfere with membrane stability. In order to use this part you can produce it in a TAG recoded organism simultaneously expressing a Tyrosine supressor or L-DOPA orthogonal translational system. In order to purify you can use the 2X Strep tag and strep column and later cleave with enterokinase to remove the sequence supressing LL-37 antimicrobial action.

LL-37-MFP: BBa_K1396001

The part is an coding sequence for an anti-microbial peptides linked to a mussel-foot protein. The mussel foot protein will anneal to surfaces as a wet glue and the antimicrobial domain is designed to interact with microbial membranes and interfere with membrane stability. In order to use this part you can produce it in a TAG recoded organism simultaneously expressing a Tyrosine suppressor or L-DOPA orthogonal translational system. In order to purify you can use the 2X Strep tag and strep column and later cleave with enterokinase to remove the sequence suppressing LL-37 antimicrobial action. This is an improvement on the Utah State biobrick BBa_K1162006 which consists of only the LL-37 peptide.

MFP-sfGFP: BBa_K1396002

The part is an coding sequence for an anti-microbial peptides linked to a mussel-foot protein-linked to superfolder GFP for localization. The mussel foot protein will anneal to surfaces as a wet glue and superfolder GFP will allow for florescence imaging and localization. In order to use this part you can produce it in a TAG recoded organism simultaneously expressing a Tyrosine supressor or L-DOPA orthogonal translational system. In order to purify you can use the 2X Strep tag and strep column and later cleave with enterokinase to remove the sequence supressing LL-37 antimicrobial action.

Mussel Foot Protein 1-5-1: BBa_K1396003

Recoded and codon optimized coding sequence for the mussel foot protein 151. TAG is recoded. In order to produce the protein co-express in cells contain either an L-DOPA orthogonal translation system or a Tyrosine suppressor.

Main Campus:
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Phone: 203.432.3783
igem@yale.edu
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Copyright (c) 2014 Yale IGEM