Team:IISER-Bhopal-India/Integrated Human Practices
Introduction
Integrated Human Practices was a continuous and meaningful way for our team to take into account public and expert opinions. Assimilation of such inputs and feedbacks helped us assess the viability of our experimental approach and project design. Our team aimed at addressing two major concerns with respect to our project:
Safety and Environmental Impact of MethNote.
Directed Product Design by implementing inputs of Experts and Stakeholders.
Safety and Environmental Impact
Recombinant DNA technology is the foundation of SynBio and is extensively used to explore myriad of biological questions. Upon discussing its limitations, we came across more poignant issues of Antibiotic Resistance (ABR) and multidrug resistance conferred to GMOs during the process.
It is important to consider that we are creating a proof-of-concept rather than an immediately implementable product. Thus, safety concerns involved at this stage mainly revolved around developing a recombinant Pichia pastoris which was resistant to multiple (precisely, 3) antibiotics.
Discussion of our initial project strategy with Dhananjay Kumar, IISER’s alumnus, raised concerns about multi-drug resistant yeast strain that might turn hazardous in case of accidental release. Working upon his inputs, we brainstormed and looked through the experiences of past iGEM teams and remodeled our wet-lab strategy by introducing a viral peptide 2A system. These peptides are about 20 amino acids long and are inserted between 2 genes in a eukaryotic system. 2A peptides have the potential of reducing the current number of required plasmids, hence reducing the number of selectable markers, used in our experimental approach. The beauty of this system is that no separate promoters or terminators are required for expression of each gene. Following this discussion we explored more and developed our alternate strategy which led to the construction of two new parts (BBa_K2790004, BBa_K2790005) to the Registry.
Further discussion with Dr. Himanshu Kumar, Associate Professor at the Department of Biological Sciences, introduced us to Cre-Lox Recombination. It is a type of site-specific recombination that could be used to delete loxP-flanked DNA. Our future work strategy involves flanking each antibiotic resistance gene with LoxP sites and introducing the Cre recombinase under an inducible promoter. Upon addition of appropriate inducer, the translated Cre enzyme would delete the floxed DNA making our recombinant Pichia pastoris sensitive to the antibiotics present in its growth media. The Cre-Lox system basically would act as a kill-switch in case our recombinant chassis organism becomes hazardous for the environment.
Directed Product Design by implementing inputs of Experts and Stakeholders.
Whilst we made modifications to our designs in the lab, a visit to a Maruti Gas Cylinders- a local cylinder manufacturing industry made us realize that the users choose not to rely on the existing sensing-methods to detect gas leakages because of lack of sensitivity. An improvement seemed imperative for the same. The future prospect of implementing our prototype in godowns also came into the picture from this visit.
Our second visit aimed to evaluate the need for a biosensor in industries which have discontinued the use of other gas-based sensors. On visiting Rail Spring Factory, we learned that gas clouding is vital for the detection of fugitive emissions. As MethNote currently is a laboratory-based prototype for a robust field-based biosensor, our future plans are directed towards addressing these concerns while product designing.
Specificity of the gas detection is a major concern in industries.Thanks to SynBio approaches,our bioengineered yeast, Pichia pastoris is designed for the specific detection of methane hence it stands as one of the ideal solutions to check for fugitive emissions.
One of our experts’ interview with Dr. Dhanyalekshmi Pillai- Assistant Professor at the Department of Earth and Environmental Sciences helped us view our prototype from the perspective of a potential buyer. She suggested that, in order to compete with pre-existing biosensors in the market, MethNote should be cost-effective, and allow in-situ detection of methane
She suggested that in-situ measurements have more precision than satellite imaging, thus measuring the local concentration of methane can be considered as a future application of MethNote, especially for monitoring deep areas/ pipelines.Along with the detection of fugitive emissions, other possibilities of our prototype can be explored, such as the production of biofuel methanol, that has 1000 times more energy density than that of methane and can be easily stored as well.
Since our project revolved around recombinant P. pastoris, we got in touch with one of Dr. Lavanya’s colleagues -Dr. Amit Verma- A senior scientist from Indian Institute of Toxicological Research, Lucknow who has been working with the yeast. He gave us a view of the possible issues that one might face while working with P. pastoris. He urged us to optimize MethNote to be temperature-tolerant for it to be successfully useful in industrial spaces. Dr. Amit suggested using colorimetric assay to detect methanol ( intermediate in our proposed pathway). We plan to incorporate all the valuable suggestions into the product designing our prototype.
