Team:Nevada

Pyruvate Decarboxylase & Alcohol Dehydrogenase coding regions under Sigma 70 Constitutive Promoter (J23101)
We first tested ethanol production in s70/PDC/ADH in NEB B10 E. coli cells using EnzyChrom Ethanol Detection Kit (BioAssay Systems). 20 samples were tested, however no samples yielded absorbance over background.

An ADH enzymatic assay was performed by the Enzymology team. Results were negative as ethanol standards of low concentrations (<20µM) were not detected. Only when high concentrations of ethanol were added was there any absorbance correlating to ADH activity. We concluded that the ADH enzyme was not sensitive enough to detect ethanol within the concentration ranges we were expecting, and opt to use a more sensitive Alcohol Oxidase enzyme instead.

To test the presence of PDC activity, Aldehyde Detection plates were created. When an aldehyde is present, a bright pink to red colony will form after 2 hours of incubation. (insert original picture from article here). After streaking s70/PDC/ADH in NEB B10 E. coli cells onto the plates, we yielded pink colonies after 2 hours of incubation which intensified after overnight incubation (insert s70/PDC/ADH in NEB B10 picture).

To once again test ADH activity, we transformed s70/PDC/ADH into NEB High Expression Iq cells. These cells were plated on the aldehyde detection plates and demonstrated a more intense pink colony than the NEB B10 cells. (insert s70/PDC/ADH in NEB Iq picture).

The EnzyChrom Ethanol Detection Kit to test ethanol production in s70/PDC/ADH in NEB Iq E. coli cells. Here, we grew the cultures with the addition of 2% glucose, since glucose can help improve E. coli growth. With these constructs, we were able to detect 0.02% Ethanol. (insert graph)

To further improve ethanol production, we contacted UniPavia, who in 2009 was able to attain 3% ethanol production. They used 10% glucose and induced cultures with 1% ethanol (to improve ADH activity).