Team:Missouri Miners/Project


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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.

MS&T Methanol Sensor Team

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.

MS&T Bio-Battery Team

Overall project

The goal of this research is the manipulation of yeast cells; granting them the capability of measuring the concentration of ethanol present. This project utilizes the metabolic pathways of the yeast Pichia pastoris, which are capable of metabolizing ethanol and methanol. The enzyme, alcohol oxidase (AO), encoded in the AOXI gene appears to be the major enzyme involved in methanol metabolism. If both carbon sources are present, however, P. pastoris prefers to utilize ethanol first. This preference is controlled by the AOXI promoter. Fusing the AOXI promoter with a fluorescent protein gene will allow visible detection of the expression of AOXI. In supplying the yeast with ethanol and methanol simultaneously, the cells should produce the fluorescent protein after ethanol consumption; resulting in a visible color and fluorescence. The concentration of ethanol can be determined by measuring the time before fluorescence and in doing so, will make plausible the development of a breathalyzer device and additional sensor systems.

Project Details


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.


Possible Methanol Sensor Applications:

  • Gasoline: Both methanol and ethanol are used to alter the oxygen content of gasoline, creating the need for a way to detect and measure these additives.
  • Homebrewing: It's well known that if you don't distill the methanol out of your moonshine you'll go blind. A bacteria-based biological methanol-sensor could provide an inexpensive way to test the safety of a distillate.
  • Methanol Fermentation: There are a few species of bacteria and yeast that use methanol as a fermentation substrate. Too much however will disrupt the metabolic process.