Team:Missouri Miners/Project

Methanol Sensor:

The purpose of this research is to use recombinant technology to culture yeast cells capable of determining the concentration of ethanol and using these cells to construct an ethanol sensor. Metabolic pathways exist for the metabolism of methanol and ethanol within some species of the Pichia taxa to include the yeast of our interest, Pichia pastoris. Alcohol oxidase (AO) appears to be the first and major enzyme produced in the methanol metabolic pathway of P. pastoris. However, if both ethanol and methanol are present, P. pastoris will utilize the ethanol before consuming the methanol. Consequently, the AOXI gene will not be expressed to produce the AO enzyme until the ethanol has been consumed.

Fusing the AOXI gene promoter with the DNA sequence encoding a fluorescent protein will allow the expression of the AOXI gene to be detected. In supplying the yeast cells with ethanol and methanol simultaneously, the cells will produce the fluorescent protein once the ethanol is utilized. The concentration of ethanol can then be determined by measuring the time before fluorescence is detected.

FUTURE PROJECT - Microbial Fuel Cell:

Optimization of electron shuffle to external surfaces such as anodes is a primary goal. Geobacter sulfurreducens happens to be our model bacteria due to its ability in nature to efficiently export electrons extracelluarly. E. coli can be the chassis for this experiment due to its genome already containing some key proteins in our preferred pathway. The proteins, such as extracellular pilin, MacA, and many other cytochromes, which E. coli does not have will be isolated from Geobacter sulfurreducens and introduced into E. coli to formulate the most optimal pathway for generating electronmotive force in a microbial fuel cell apparatus.

Some problems will be faced concerning plasmid engineering and the simple fact that Geobacter is anaerobic and E. coli is aerobic. As a team, we will push in the right direction harder than an emf on the internal resistivity of a toroid. Many diverse team members will work in concert utilizing Missouri S&T’s dominating Electrical, Chemical, and Biological Engineering undergraduates along with Biological Science masterminds.

Some of the techniques that have been used thus far in pursuing this project have been:

1. PCR amplification of the AOX1 promoter using PCR primers that added standard prefix and suffix sequences
2. Cloning the AOX1 PCR product into a commercial cloning vector
3. Digesting the AOX1, RFP and GFP (later unused) plasmids to obtain the promoter and reporter genes of interest
4. Separating and isolating the enzyme digest fragments through gel electrophoresis
5. Ligating the AOX1 promoter fragment in the RFP plasmid
6. Transforming bacterial cells with the recombined AOX/RFP plasmid

Additional steps which will be taken in the continuance of this project:

1. Dephosphorylating fragments during ligation to ensure greater chance of successful recombination
2. Reintroducing the recombinant DNA to Pichia pastoris if Escherichia coli is unable to express RFP from the AOX1
3. This will require cloning the AOX1/RFP fusion into a yeast vector

If a proof of concept of the expression of RFP under the control of the AOX1 promoter can be obtained, then the next step will be to design a system for the introduction of methanol/ethanol to the organism in a way that can be measured quantitatively.