Difference between revisions of "Team:Aalto-Helsinki/LabResults"

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<h3>Overview</h3>
 
<h3>Overview</h3>
  
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<p>Our goal was to produce propane from cellulose in <i>E.coli</i>. To achieve this our aim was to build three plasmids and transform them to the same bacteria. Two of the plasmids include the ten genes necessary for propane production in <i>E.coli</i>. The third plasmid contains the three genes encoding the enzymes for cellulose hydrolysis.
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<p>Our goal was to produce propane from cellulose in <i>E.coli</i>. To achieve this our aim was to build three plasmids and transform them to the same bacteria. Two of the plasmids, Propane 1 and Propane 2 include the ten genes necessary for propane production in <i>E.coli</i>. The third plasmid contains the three genes encoding the enzymes for cellulose hydrolysis. When choosing the backbones for Propane plasmid 1, Propane plasmid 2 and Cellulose plasmid we took into account that the backbones have intercompatible origins of replication.
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To ease the bottlenecks caused by two of the enzymes in the propane pathway we built an amphiphilic protein biobrick. To validate our amphiphilic protein we created a new, fusable GFP biobrick. Amphiphilic micelle formation was validated with electron microscopy.</p>
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In the lab we created three Biobrics: Propane plasmid 1, Fusable GFP and Amphiphilic protein. Check the full information about and laboratory results of the Biobrics here.</p>
  
 
<h3>Propane producing plasmid 1: CAR</h3>
 
<h3>Propane producing plasmid 1: CAR</h3>
  
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<p>CAR is the first of our propane producing plasmids. The plasmid was constructed to pSB6C1 backbone with Gibson assembly. Afterwards the CAR insert was transferred to the biobrick backbone pSB1C3 with restriction digestion and ligation.</p>
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<p>CAR is the first of our propane producing plasmids. The plasmid was constructed to pSB6C1 backbone with Nebuilder assembly. Afterwards the CAR insert was transferred to the biobrick backbone pSB1C3 with restriction digestion and ligation.</p>
  
 
<p>Achievements:</p>
 
<p>Achievements:</p>
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<p> - Gibson assembly with pSB6A1 backbone successful according  to colony-PCR</p>
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<p> - NeBuilder assembly with pSB6A1 backbone successful according  to colony-PCR</p>
 
<p> - Successfully transferred CAR construct to biobrick backbone pSB1C3</p>
 
<p> - Successfully transferred CAR construct to biobrick backbone pSB1C3</p>
  
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<h3>Propane producing plasmid 2: AtoB</h3>
 
  
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<p>AtoB is the second of our propane producing plasmids. The plasmid was straight built to the biobrick backbone pSB1C3 with Gibson assembly.</p>
 
  
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<p>Achievements:</p>
 
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<p> - Gibson assembly with biobrick backbone pSB1C3 successful according to restriction analysis</p>
 
  
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<h3>Amphiphilic protein</h3>
 
  
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<p>Ampihilic protein was created to fuse the two bottleneck enzymes of our propane pathway close to each other.</p>
 
  
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<p>Achievements:</p>
 
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<p> - Amphiphilic protein biobrick was sequenced successfully</p>
 
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<h3>Fusable GFP</h3>
 
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<p>To help testing our amphiphilic protein we built a fusable GFP biobrick. The fusable GFP biobrick has one additional nucleotiode prior to it’s suffix: it can be fused with any aminoacid’s amino terminus without losing it’s reading frame.</p>
 
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<p>Achievements:</p>
 
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<p> - Created a fusable GFP biobrick</p>
 
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<p> - Fusable GFP was sequenced successfully</p>
 
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<p> - Successfully fused the fusable GFP biobrick with ampiphilic protein</p>
 
  
 
</section>
 
</section>

Revision as of 11:11, 17 September 2015

Laboratory Results

The constructs

Overview

Our goal was to produce propane from cellulose in E.coli. To achieve this our aim was to build three plasmids and transform them to the same bacteria. Two of the plasmids, Propane 1 and Propane 2 include the ten genes necessary for propane production in E.coli. The third plasmid contains the three genes encoding the enzymes for cellulose hydrolysis. When choosing the backbones for Propane plasmid 1, Propane plasmid 2 and Cellulose plasmid we took into account that the backbones have intercompatible origins of replication. In the lab we created three Biobrics: Propane plasmid 1, Fusable GFP and Amphiphilic protein. Check the full information about and laboratory results of the Biobrics here.

Propane producing plasmid 1: CAR

CAR is the first of our propane producing plasmids. The plasmid was constructed to pSB6C1 backbone with Nebuilder assembly. Afterwards the CAR insert was transferred to the biobrick backbone pSB1C3 with restriction digestion and ligation.

Achievements:

- NeBuilder assembly with pSB6A1 backbone successful according to colony-PCR

- Successfully transferred CAR construct to biobrick backbone pSB1C3