Team:IISER-Bhopal-India/Prospects
Future Prospects
In our project MethNote, we have aimed to develop a genetically modified yeast, Pichia pastoris in which genes coding for an enzyme from a methylotrophic bacteria M. capsulatus have been incorporated. This enzyme, sMMO (soluble Methane Monoxygenase) catalyzes the conversion of methane to methanol, which is not an indigenous reaction present in Pichia pastoris.
The current project is a proof-of-concept project that aims to check the expression of the enzyme subunits in our chassis Pichia pastoris. Further thoughts about improvement in our cloning strategy that could potentially eliminate the use of antibiotic markers to prevent the introduction of multidrug resistance in Pichia are underway.
Once this GMO is developed, it can have a variety of future applications in various fields. Ranging from our initial thoughts of developing a prototype for a Methane Biosensor to providing an important substrate for Biofuel production and methane sequestration could be achieved by further optimization and modelling of the project as a whole. We would like to develop a practically applicable product that could help address environmental issues and have industrial applications. After iGEM, our project could be a potential start-up under the umbrella of IICE (Innovation and Incubation Centre for Entrepreneurship), IISER Bhopal.
Optimised Cloning Strategy
Multi-drug resistance in genetically modified microorganisms is rapidly becoming a huge health hazard in many parts of the world. Keeping this in mind we decided to reduce the number of antibiotic markers used in our cloning strategy. After having a talk with one of our professors, we got the insight for using a Cre-LoxP system for eliminating resistance markers altogether from our GMO. We planned to clone the subunits for sMMO into engineered plasmids carrying the resistance markers flanked with LoxP sites in opposite orientations. After transformation, these plasmids would integrate into the host chromosomal DNA and then the inducible plasmid carrying the Cre-recombinase would be introduced into the host. After Cre expression, the antibiotic resistance genes would be excised and the organism would be free of antibiotic resistance while being able to stably express the sMMO subunits.
Methanol Production
The microbial route of methanol biosynthesis first involves oxidation of methane to methanol through the sMMO complex. This process is energy dependent and uses NADH. The methanol gets further oxidized to formaldehyde by NADH-independent methanol oxidase/dehydrogenase activity.
We could potentially optimize this biosensor to sequester large amounts of methane from the environment. Controlled release of these organisms into strategic locations could be our small step towards curbing climate change. The device could also be used to harvest substantial amounts of methanol which could be used as a biofuel. Methanol is more energy dense and less hazardous than methane and hence is a better biofuel for future use.
Optimised Methane Sensing
Our prototype would be optimized to increase its range and sensitivity of methane sensing, such as by sensing other secondary metabolites by Colorimetric Assay, so that it could be more reliably used in industries and to better monitor changes in local methane concentrations over a period of time.