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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