Team:Arizona State/parts

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<td width="90%" ><p><strong style="font-size: 34px">The Arizona State University iGEM team</strong></p>
<td width="90%" ><p><strong style="font-size: 34px">The Arizona State University iGEM team</strong></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></td>
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   <p>&nbsp;</p>
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  <p><strong>Ethanol Production Parts</strong> <br>
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    To optimize ethanol production in our EtOH strain, we wanted to overexspress the pyruvate decarboxylase (pdc) and alcohol dehydrogenase B(adhB) enzymes in order to maximize the amount of ethanol being produced. The iGEM registry contained the part BBa_k1122676, made by Edinburgh in 2013. This part was compared to another plasmid containing pdc/adhB, donated by Dr. David Neilsen. The results indiciated that the BBa_k1122676 was the better ethanol producer, making it the key component of the EtOH strain. Registry page:
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<a href="http://parts.igem.org/Part:BBa_K1122676">BBa_K1122676</a></p>
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  <p>&nbsp;</p>
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  <hr>
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  <p>&nbsp;</p>
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  <p><strong>Fatty Acid Production Parts</strong> <br>
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    In order to maximize the amount of fatty acetyl-coenzyme A molecules being produced in the fatty acid strain, two enzymes were targeted for overproduction. The first was the thioesterase A (TesA) protein, which takes the fatty Acyl-ACP produced from glycolysis and produces free fatty acid chains. The part BBa_K654058 codes for the TesA protein, but it did not have the required components for controllable expression. In order to do this, a promoter and an RBS were planned to be added upstream of this coding sequence. The RBS was successfully inserted, but we were unable to insert the promoter in time for the part submission. 
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Part Submitted:
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<a href="http://parts.igem.org/Part:BBa_K1502000">BBa_K1502000</a></p>
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<p> The second part
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This enzyme is coded through the four acc genes, labeled accA through accD. This parts were isolated through PCR replication using different primers. accA and accB were ready for part submission, but the accC part had two Pst1 sites within its sequence and the accD sequence contained an EcoR1 site. By using gene splicing thorough overlapping sequences (SOEing), the EcoR1 site on accD was removed. This means that the accA, accB, and accD parts were submitted to the registry. </p>
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<p>
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Parts Submitted:
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<a href="http://parts.igem.org/Part:BBa_K1502001">BBa_K1502001 (aacA)</a></p>
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<a href="http://parts.igem.org/Part:BBa_K1502002">BBa_K1502002 (accB)</a></p>
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<a href="http://parts.igem.org/Part:BBa_K1502003">BBa_K1502003 (accD)</a></p>
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  <p>&nbsp;</p>
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<img src="https://static.igem.org/mediawiki/2014/4/40/ASU_acc_plasmid_image.png" alt="" align="center" height="400" >
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  <p>&nbsp;</p>
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  <p><strong>Biodesiel Synthesis Parts</strong> <br>
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    In order to combine the ethanol and the fatty acetyl-coenzyme A molecules into the desired fatty ethyl esters, a wax esterase is required. This wax esterase is coded through the atfA gene, which is found on the pKS104 plasmid. This plasmid would be inserted into either strain, and produce the desired fatty ethyl ester compound to be used as biodiesel when both components are avalible. This plasmid was ordered from Addgene, and we were unable to convert it into a part in time for submission to the registry.   </p>
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Latest revision as of 03:25, 18 October 2014

 

 

The Arizona State University iGEM team

 

Ethanol Production Parts
To optimize ethanol production in our EtOH strain, we wanted to overexspress the pyruvate decarboxylase (pdc) and alcohol dehydrogenase B(adhB) enzymes in order to maximize the amount of ethanol being produced. The iGEM registry contained the part BBa_k1122676, made by Edinburgh in 2013. This part was compared to another plasmid containing pdc/adhB, donated by Dr. David Neilsen. The results indiciated that the BBa_k1122676 was the better ethanol producer, making it the key component of the EtOH strain. Registry page: BBa_K1122676

 

 

Fatty Acid Production Parts
In order to maximize the amount of fatty acetyl-coenzyme A molecules being produced in the fatty acid strain, two enzymes were targeted for overproduction. The first was the thioesterase A (TesA) protein, which takes the fatty Acyl-ACP produced from glycolysis and produces free fatty acid chains. The part BBa_K654058 codes for the TesA protein, but it did not have the required components for controllable expression. In order to do this, a promoter and an RBS were planned to be added upstream of this coding sequence. The RBS was successfully inserted, but we were unable to insert the promoter in time for the part submission.

Part Submitted: BBa_K1502000

The second part This enzyme is coded through the four acc genes, labeled accA through accD. This parts were isolated through PCR replication using different primers. accA and accB were ready for part submission, but the accC part had two Pst1 sites within its sequence and the accD sequence contained an EcoR1 site. By using gene splicing thorough overlapping sequences (SOEing), the EcoR1 site on accD was removed. This means that the accA, accB, and accD parts were submitted to the registry.

Parts Submitted: BBa_K1502001 (aacA)

BBa_K1502002 (accB)

BBa_K1502003 (accD)

 

 

Biodesiel Synthesis Parts
In order to combine the ethanol and the fatty acetyl-coenzyme A molecules into the desired fatty ethyl esters, a wax esterase is required. This wax esterase is coded through the atfA gene, which is found on the pKS104 plasmid. This plasmid would be inserted into either strain, and produce the desired fatty ethyl ester compound to be used as biodiesel when both components are avalible. This plasmid was ordered from Addgene, and we were unable to convert it into a part in time for submission to the registry.

 

 

  

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