Team:Arizona State/notebook

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  <li><a href="https://2014.igem.org/Team:Arizona_State">home </a></li>
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  <li><a href="https://2014.igem.org/Team:Arizona_State/team">team </a>
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      <ul><li><a href="https://2014.igem.org/Team:Arizona_State/members">members </a></li>
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  <li><a href="https://2014.igem.org/Team:Arizona_State/project">project </a>
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  <li><a href="https://2014.igem.org/Team:Arizona_State/parts">parts </a></li>
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  <li><a href="https://2014.igem.org/Team:Arizona_State/result">results </a></li>
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  <li><a href="https://2014.igem.org/Team:Arizona_State/notebook">notebook </a></li>
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  <li><a href="https://2014.igem.org/Team:Arizona_State/safety">safety </a></li>
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<a href="https://2014.igem.org/Team:Arizona_State/notebook"style="color:#ffffff">notebook </a> </td>
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<td width="90%" ><p><strong style="font-size: 34px">Notebook</strong></p>
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  <hr>
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  <p>&nbsp;</p>
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  <p><strong>Project Phases</strong> <br>
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<p>&nbsp;</p>
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    <p>June: Project planning and set up</p>
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<p>July-October: Synthesis of Fatty acid producing plasmids, such as the TesA plasmid</p>
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<p>September - October: Preparation and testing of the ethanol producing parts</p>
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<p>October: Preparation and testing of the wax esterase plasmid</p>
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<p>The Arizona State University iGEM team has taken on a project of creating biodiesel in a new and efficient way using Escherichia coli. Using a wax esterase, fatty acids and ethanol, which are both naturally produced by E. coli, can be combined to form biodiesel. However, E. coli uses the same intermediate products to produce both fatty acids and ethanol, which reduces the efficiency of this process. This problem can be avoided by engineering two different strains of E. coli to work in tandem. One strain will be focused on producing free fatty acids (FFA) through increased production of enzymes such as TesA and acc. The other strain will be focused on producing ethanol through the increased production of enzymes such as pdc and adhB. These two products can be combined in one of the two cells using a waxy esterase, producing the final biodiesel product. This form of cooperative production will hopefully produce more ethanol than current single strain methods using E. coli.</p>
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  <p><strong>Procedures</strong> <br>
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    Gel making/running: Gels were all 1% agarose gels using safe white dye. Al were ran for 1 hr. at 95 V.
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<p>
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    Enyzme digestions/ Ligations: Our team had a bit of a learning curve with digestions and ligations. We tried several different methods to ensure they would work, but the most common was the procedure found in the <a href="http://openwetware.org/wiki/Haynes:Assembly101">Haynes Lab Procedures</a>
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<p>&nbsp;</p>
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  <p><strong>Resting Cell Assay</strong> <p>
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    To test the productivity of different plasmids, resting cell assays were run. All assays were run in a shaker incubator at 32 degrees C and 200 RPM. A phosphate buffered solution was used to suspend the growth of the cultures.  </p>
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<p>&nbsp;</p>
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  <p><strong>Quantification</strong> <p>
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A GC with a DB5 column was used to quantify data from all assays. For testing ethanol production, the method used had an initial temp. of 60 degrees C, and ramped by 10 degrees C until it reached 250 degrees C, which was then held for 3 minutes.
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Latest revision as of 03:08, 18 October 2014

 

 

Notebook

 

Project Phases

 

June: Project planning and set up

July-October: Synthesis of Fatty acid producing plasmids, such as the TesA plasmid

September - October: Preparation and testing of the ethanol producing parts

October: Preparation and testing of the wax esterase plasmid

 

 

Procedures
Gel making/running: Gels were all 1% agarose gels using safe white dye. Al were ran for 1 hr. at 95 V.

Enyzme digestions/ Ligations: Our team had a bit of a learning curve with digestions and ligations. We tried several different methods to ensure they would work, but the most common was the procedure found in the Haynes Lab Procedures

 

Resting Cell Assay

To test the productivity of different plasmids, resting cell assays were run. All assays were run in a shaker incubator at 32 degrees C and 200 RPM. A phosphate buffered solution was used to suspend the growth of the cultures.

 

Quantification

A GC with a DB5 column was used to quantify data from all assays. For testing ethanol production, the method used had an initial temp. of 60 degrees C, and ramped by 10 degrees C until it reached 250 degrees C, which was then held for 3 minutes.

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