Team:Yale/Parts

From 2014.igem.org

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<tr><td colspan="4"><h2>Modeling E. coli growth producing a toxic compound</h2>
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<tr><td colspan="4"><h2>Submitted Parts</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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The collection consists of the BioBrick mussel foot protein (MFP) 1-5-1 sequence (combination of  Mytilus galloprovincialis Foot Protein 5 (Mgfp-5) and Mytilus Edulis Foot Protein1 (Mefp-1)). The second BioBrick is the MFP with superfolder Green Fluorescence Protein (sfGFP). The third BioBrick is the MFP with our antimicrobial peptide of interest, LL-37. Finally, the fourth BioBrick is our entire construct of our antimicrobial peptide: 2XStrep_Flagtag_LL-37_Mussel Foot Protein_sfGFP. All biobricks are in the pSB1C3 plasmid.
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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>Determining Optimal Time to Induce Expression</h2>
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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></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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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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<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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Revision as of 02:14, 17 October 2014

Parts

Submitted Parts

The collection consists of the BioBrick mussel foot protein (MFP) 1-5-1 sequence (combination of Mytilus galloprovincialis Foot Protein 5 (Mgfp-5) and Mytilus Edulis Foot Protein1 (Mefp-1)). The second BioBrick is the MFP with superfolder Green Fluorescence Protein (sfGFP). The third BioBrick is the MFP with our antimicrobial peptide of interest, LL-37. Finally, the fourth BioBrick is our entire construct of our antimicrobial peptide: 2XStrep_Flagtag_LL-37_Mussel Foot Protein_sfGFP. 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.

Main Campus:
Molecular, Cellular & Developmental Biology
219 Prospect Street
P.O. Box 208103
New Haven, CT 06520
Phone: 203.432.3783
igem@yale.edu
natalie.ma@yale.edu (Graduate Advisor)
Copyright (c) 2014 Yale IGEM

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