Part:BBa_K558001
s70/PDC/ADH Ethanol Generator
This part includes the coding regions for Pyruvate Decarboxylase (PDC) and Alcohol Dehydrogenase (ADH) based on Zymomonas mobilis along with a standard E. coli ribosome binding site and double terminator(BBa_B0014). Under regulation by the Sigma 70 constitutive promoter (iGEM part J23101), this generator may be used for Ethanol Production.
Pyruvate Decarboxylase Activity
Pyruvate decarboxylase activity was assayed using aldehyde indicator plates (Conway et. al. 1987) to test for a Schiff Base reaction to detect the presence of acetaldehyde, a substrate for ethanol production. The figure below shows pink colonies after an overnight incubation on Schiff base plates. The streaked colony on the top of the plate is a negative control cell line (white-light pink). The streaked colony on the right is a typical example (pink color) of sigma 70-pdc/adh generator in the NEB 10-Beta cells. The cells on the left (dark pink) represent what is typically seen for the sigma 70-pdc/adh generator expressed in T7 Express Iq cell lines. From the results shown here and other experiments, we concluded that the NEB T7 Express Iq cells produced higher levels of the acetaldehyde intermediate.
The pyruvate decarboxylase gene converts pyruvate to acetaldehyde. Acetaldehyde is then converted to ethanol by alcohol dehydrogenase.
Alcohol Dehydrogenase Activity
Ethanol production was tested using EnzyChrom Ethanol Assay Kit (BioAssay Systems). The figure below shows percent ethanol production in T7 Express Iq cells (NEB) in 2% versus 10% glucose medium over 24 and 48 hours. The sample grown in 2% Glucose over 24 hours yielded 0.02% ethanol, and the sample grown in 10% Glucose over 48 hours yielded 0.018% ethanol. From the results shown here, and other experiments, we have concluded that our modified E. coli is in fact producing ethanol. Furthermore, we have also concluded that increasing amounts of glucose does not lead to higher ethanol production. It is possible that these lines are only producing low levels of alcohol dehydrogenase and we are seeing a buildup of acetaldehyde in these same lines. It may be necessary to re-evaluate the original operon and re-engineer it to improve the adh gene thereby increasing production of the alcohol dehydrogenase enzyme.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NotI site found at 1762 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1770
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1066
Illegal AgeI site found at 1213
Illegal AgeI site found at 1591
Illegal AgeI site found at 2362 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 100
Illegal BsaI site found at 2713
Illegal SapI site found at 514
Illegal SapI site found at 1177
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