Team:Northwestern/methods

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<div class = "container">
 
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<div class = "menu">
 
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<ul>
 
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<li><a href="https://2013.igem.org/Team:Northwestern">Home</a></li>
 
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<li><a href="https://2013.igem.org/Team:Northwestern/Team">Team</a>
 
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<ul>
 
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<li><a href="https://igem.org/Team.cgi?year=2013&team_name=Northwestern">Official Profile</a></li>
 
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<li><a href="#">Meet Us!</a></li>
 
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<li><a href="https://2013.igem.org/Team:Northwestern/Attributions">Attribution</a></li>
 
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</ul>
 
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</li>
 
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<li><a href="https://2013.igem.org/Team:Northwestern/Project">Project</a>
 
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<ul>
 
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<li><a href="https://2013.igem.org/Team:Northwestern/Parts">Parts Submitted</a></li>
 
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<li><a href="https://2013.igem.org/Team:Northwestern/Notebook">Notebook</a></li>
 
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<li><a href="#">Description</a></li>
 
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</li>
 
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<li><a href="https://2013.igem.org/Team:Northwestern/Modeling">Modeling</a></li>
 
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<li><a href= "https://2013.igem.org/Team:Northwestern/Safety"> Safety </a></li>
 
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</br>
<div>
<div>
<h1> Methods Overview </h1>
<h1> Methods Overview </h1>
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Different Methods will be presented below.
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<center> <img src="https://static.igem.org/mediawiki/2013/6/6d/Dual-state.png"/> </center>
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<p>Our dual-state construct consists of a pH-inducible promoter, joined in series with a nonsense spacer region, a constitutive promoter, a ribosomal binding site, and a green-fluorescent protein capped with a terminator, as diagrammed above.</p>
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<p>In creating our dual-state constructs, we worked with a low-copy plasmid, plasmid pSB4A5 taken from iGEM 2013’s distribution kit. This plasmid was taken from Kit Plate 5, well 21F. We wanted to ensure that our engineered cells would survive with our constructs, as they would need to survive should our construct be implemented as intended. We used a low-copy plasmid, because a high-copy plasmid would put too much stress on our cells and hinder their survival.</p>
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<p>The table below summarizes the combinations of pH-inducible promoters, spacers, and constitutive promoters we attempted to construct into dual-state promoters.</p>
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<center> <img src="https://static.igem.org/mediawiki/2013/4/4a/Dual_state_table.png"/> </center>
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<center> <p><b>The diagrams below demonstrate our cloning strategy in creating these dual-state promoters.<b></p> </center>
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<p><b>Stage 1: </b> We began by creating constructs containing Asr-rbs, GadA-rbs, Lpp-RBS, and TacRBS in conjunction with GFP and a terminator sequence in order to test the fluorescence of individual promoters at different pH levels (pH 3.5, 4.5, 5.5, 6.5, 7.5). This allows us to compare activities of these promoters in the dual-state against their activities in the single-state to better assess how these promoters might operate differently in a dual-state.</p>
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<center> <img src="https://static.igem.org/mediawiki/2013/e/ef/Diagram1.png" height="400" width="500"/></center>
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<p><b>Stage 2a: </b> We proceeded by placing each of our pH-inducible promoters upstream of the each of our nonsense spacer regions.</p>
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<center><img src="https://static.igem.org/mediawiki/2013/a/af/Diagram2.png" height="350" width="500" /></center>
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Stage 2b: Finally, we were able to construct our dual-state promoters. We placed our pH-inducible promoter+spacer region constructs upstream constitutive promoter+RBS+GFP+terminator constructs. We were able to obtain these constitutive promoter+RBS+GFP+terminator constructs from work our TA Jessica Perez did separately as a member of the Jewett Laboratory at Northwestern University. We characterized these constructs by performing a fluorescence assay with minimal growth media buffered to pH 3.5, 4.5, 5.5, 6.5, and 7.5.</p>
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<center><img src="https://static.igem.org/mediawiki/2013/e/e2/Diagram3.png" height="400" width="400" /> </center>
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</div>
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<h1>Strains and Media</h1>
<h1>Strains and Media</h1>
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       <p>scherichia coli Top10 (Invitrogen) was used for all transformations and assays. Media included SOB for transformations and LB for overnight cultures. Transformed strains were grown at 37°C using Ampicillin resistance. Primers for PCR were purchased from Integrated DNA Technologies (IDT) and New England Biolabs (NEB) donated all of the restriction enzymes. All sequencing was conducted by the Northwestern Genomics Core.</p>  
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       <p>Escherichia coli Top10 (Invitrogen) was used for all transformations and assays. Media included SOB for transformations and LB for overnight cultures. Transformed strains were grown at 37°C using Ampicillin resistance. Primers for PCR were purchased from Integrated DNA Technologies (IDT) and New England Biolabs (NEB) donated all of the restriction enzymes. All sequencing was conducted by the Northwestern Genomics Core.</p>  
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    <h1>Forming a Library of Constructs </h1>
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      <div>
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          <p>e low copy plasmid pSB4A5 will be used for the different promoter constructs. The asr and gadA promoters were extracted from the E. coli genome via colony polymerase chain reaction (PCR). The constitutive promoters TacI and Lpp were amplified from pDAK1 and pDAK2 donated by the Jewett Lab6,7. The restriction enzyme cut sites EcoR1, Pst1, Spe1, and Xba1 were used in ligation to create the different constructs (Figure 3). When multiple parts were connected a mixed site was formed between Spe1 and Xba1, which cannot be cut, by any of the restriction enzymes. This leads to the benefit of not having a restriction site in the middle of the construct, and furthermore these standard restriction enzymes can always be used with the final dual-state promoter constructs.<p/>
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      <center> <img src="https://static.igem.org/mediawiki/2013/thumb/6/60/Construct_one.png/750px-Construct_one.png" height="200" width="200"/>
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          <img src="https://static.igem.org/mediawiki/2013/thumb/5/5a/Construct_two.jpg/750px-Construct_two.jpg" height="200" width="200" />
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          <img src="https://static.igem.org/mediawiki/2013/thumb/b/bb/Construct_three.jpg/750px-Construct_three.jpg" height="200" width="200" /> </center>
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<h1> Protocols </h1>
<h1> Protocols </h1>
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<div>
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<h2> Supplies </h2>
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<h2> Supplies </h2><center>
<p> <a href="https://2013.igem.org/Team:Northwestern/makem9">Making M9 Media</a></p>
<p> <a href="https://2013.igem.org/Team:Northwestern/makem9">Making M9 Media</a></p>
     <p> <a href="https://2013.igem.org/Team:Northwestern/makecells">Making Competent Cells</a></p>
     <p> <a href="https://2013.igem.org/Team:Northwestern/makecells">Making Competent Cells</a></p>
     <p> <a href="https://2013.igem.org/Team:Northwestern/transform">Transforming Competent Cells</a></p>
     <p> <a href="https://2013.igem.org/Team:Northwestern/transform">Transforming Competent Cells</a></p>
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     <p> <a href="https://2013.igem.org/Team:Northwestern/lb">Making LB Plates</a></p>
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     <p> <a href="https://2013.igem.org/Team:Northwestern/lb">Making LB Plates</a></p></center>
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<h2> Clonning </h2>
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<h2> Assay </h2><center>
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    <p> <a href="https://2013.igem.org/Team:Northwestern/fluo">Fluorescence Assay</a></p></center>
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<h2> Cloning </h2><center>
       <p> <a href="https://2013.igem.org/Team:Northwestern/primers">Annealing Primers</a></p>
       <p> <a href="https://2013.igem.org/Team:Northwestern/primers">Annealing Primers</a></p>
       <p> <a href="https://2013.igem.org/Team:Northwestern/pcr">PCR Amplification</a></p>
       <p> <a href="https://2013.igem.org/Team:Northwestern/pcr">PCR Amplification</a></p>
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       <p> <a href="https://2013.igem.org/Team:Northwestern/digest">Restriction Digest</a></p>
       <p> <a href="https://2013.igem.org/Team:Northwestern/digest">Restriction Digest</a></p>
       <p> <a href="https://2013.igem.org/Team:Northwestern/ligate">Ligation</a></p>
       <p> <a href="https://2013.igem.org/Team:Northwestern/ligate">Ligation</a></p>
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       </center>
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<h2> Assays and Etc </h2>
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    <p> <a href="https://2013.igem.org/Team:Northwestern/fluo">Fluorescence Assay</a></p>
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Latest revision as of 04:04, 28 September 2013


Methods Overview

Our dual-state construct consists of a pH-inducible promoter, joined in series with a nonsense spacer region, a constitutive promoter, a ribosomal binding site, and a green-fluorescent protein capped with a terminator, as diagrammed above.

In creating our dual-state constructs, we worked with a low-copy plasmid, plasmid pSB4A5 taken from iGEM 2013’s distribution kit. This plasmid was taken from Kit Plate 5, well 21F. We wanted to ensure that our engineered cells would survive with our constructs, as they would need to survive should our construct be implemented as intended. We used a low-copy plasmid, because a high-copy plasmid would put too much stress on our cells and hinder their survival.

The table below summarizes the combinations of pH-inducible promoters, spacers, and constitutive promoters we attempted to construct into dual-state promoters.

The diagrams below demonstrate our cloning strategy in creating these dual-state promoters.

Stage 1: We began by creating constructs containing Asr-rbs, GadA-rbs, Lpp-RBS, and TacRBS in conjunction with GFP and a terminator sequence in order to test the fluorescence of individual promoters at different pH levels (pH 3.5, 4.5, 5.5, 6.5, 7.5). This allows us to compare activities of these promoters in the dual-state against their activities in the single-state to better assess how these promoters might operate differently in a dual-state.

Stage 2a: We proceeded by placing each of our pH-inducible promoters upstream of the each of our nonsense spacer regions.

Stage 2b: Finally, we were able to construct our dual-state promoters. We placed our pH-inducible promoter+spacer region constructs upstream constitutive promoter+RBS+GFP+terminator constructs. We were able to obtain these constitutive promoter+RBS+GFP+terminator constructs from work our TA Jessica Perez did separately as a member of the Jewett Laboratory at Northwestern University. We characterized these constructs by performing a fluorescence assay with minimal growth media buffered to pH 3.5, 4.5, 5.5, 6.5, and 7.5.

Strains and Media

Escherichia coli Top10 (Invitrogen) was used for all transformations and assays. Media included SOB for transformations and LB for overnight cultures. Transformed strains were grown at 37°C using Ampicillin resistance. Primers for PCR were purchased from Integrated DNA Technologies (IDT) and New England Biolabs (NEB) donated all of the restriction enzymes. All sequencing was conducted by the Northwestern Genomics Core.

Protocols