The 2011 Wisconsin-Madison team's project

This summer we sought to produce and improve biofuel biosensors in order to optimize the biofuel discovery and refinement process. We developed and tested an ethanol sensor which used genes from Pseudomonas aeruginosa to fluoresce red in the presence of ethanol. This process is illustrated below:

After determining that, despite being codon optimized for E. coli, the system did not function at a high enough level to be easily visible, we decided to try performing a directed evolution study on our sensor to decrease basal expression and increase induced expression. We created a double selection cassette which could be used for the following directed evolution process:

The 2011 Wisconsin-Madison team's parts

- exaDE (K634002): The sensor kinase ExaD responds to the presence of ethanol within the cell and phosphorylates ExaE. The phosphorylated ExaE then acts as an activating transcription factor on PexaA.

- PexaA (K634008): This is the intergenic region 5' of exaA in Pseudomonas aeruginosa. It is the promoter in the ethanol sensor we produced.

- tagRFP (K634005): A derivative of mCherry, this fluorescent protein acts as the reporter for the ethanol sensor, as well as a component of the double selection cassette.

- Double selection cassette (K634007): This part is a composite of three parts which can be used for all steps in the above-illustrated directed evolution process.

Other important parts

- sacB (K322921): This gene, taken from Bacillus subtilis, was a crucial part of our plan for directed evolution. We discovered that though the part was characterized for use on solid media, our intended use of liquid media did not fit that same protocol. We have contributed improved information to the Experience tab of this part, in hopes that future teams will have improved results when using it in liquid media.