An early idea our team had considered was to employ cold-tolerance as a selectable marker for microbiological experiments. This was motivated by our (then) nascent understanding of antibiotic resistance as a potential threat. We considered that the near-universal employment of traditional antibiotic resistance genes in laboratory experiments would be a strong contributor to the growing incidence of drug-resistant bacterial strains. We were aiming to take antibiotic resistance completely out of the picture by bringing in cold-tolerance as a selectable resistance marker - a relatively straightforward process indeed.
Rather than the conventional protocol of cloning the gene of interest into a plasmid backbone carrying antibiotic resistance gene(s), we would develop a vector backbone with cold-tolerance conferring genes, and clone the gene of interest within this. Thereby, successful transformants would be screened out on the basis of colonies surviving under suboptimal growth conditions. Ultimately, we would be able to pluck antibiotics out of the equation, and thereby help stem the tide of drug-resistant bacterial strains!
With this in mind, we had approached professors from the Dept. of Biological Sciences, at the Indian Institute of Science, in order to obtain expert feedback about our project idea.
What followed was a true example of the IHP concept at work - and one we will always be indebted to! And while we eventually ended up dropping the entire idea of developing cold-tolerance based selection markers, we took home a number of valuable lessons in practical experimental design.
Until we had actually interacted with active researchers in this particular field and explained our concept, we had been largely unaware of the practical limitations of our idea. A brief summary of our conversations serves to illustrate the same.
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Team Members meeting Dr. Ajit Chande
Dr. Umesh Varshney, IISc Faculty
When we explained our project to Dr. Umesh Varshney, he said that it would most likely not be accepted by laboratories for a couple of reasons - the most obvious being the lack of versatility that a cold-resistant organism could offer as compared to an antibiotic-resistant one. Antibiotic resistance used as a marker allows multiple levels of selection of the organism, as opposed to a cold tolerance marker that can only provide one. Even if cold tolerance is used as a marker, it would be difficult to ensure that the colony has only transformed cells, that is, there would be no guarantee for purity of the colony. The colonies so obtained would have to be picked and plated again. Dr. Varshney also said that the cold-tolerant marker isn’t a very effective one, as it doesn’t give binary ‘yes’ or ‘no’ results. Even at 4°C, E. coli still grows, albeit at a reduced rate. So our plasmid would give a gradient of growth, which is not ideal when we’re trying to select for transformants. The results are much more definitive when antibiotic resistance is used as a marker - either the E. coli grows on the antibiotic medium, or it doesn’t. Another important point Dr. Varshney mentioned was that instead of waiting just 12 hours to check the transformants (as in when antibiotic resistance is used as a marker), we would have to wait 36 hours to get a pure colony of transformants using our method, which is not feasible at all. Also, all the work done on the transformed bacteria would have to be at a low temperature because the moment it replicates at 37℃ it will lose the plasmid, simply because there is no selection pressure on it to retain it. However, this could be countered by having an auxotrophic strain carrying an essential gene in the same plasmid as the low-temperature marker. Moreover, according to Dr. Varshney, our basic purpose for taking up this project was flawed as most antibiotic-resistant strains in the environment do not originate from labs. This is because most laboratory strains are usually mutants which would not be able to survive and grow in the wild.
Dr. Dipshika Chakravortty was of the opinion that our project would have a significant impact on antibiotic resistant (ABR) strains developing everywhere. Disposal of ABR strains has been a major issue for labs for a long time, and using alternatives like this would make disposal a lot easier and safer. She said that while the project would be helpful it’s feasibility would have to be explored further. Some factors to consider while going ahead with the project would be to check whether the plasmid is able to retain its activity on a long term basis, that is, whether multiple 4°C-37°C cycles would affect the functioning of this plasmid when considered at gaps of thousands of generations; and being able to guarantee that our method would be easy in terms of time, and precise in terms of the accuracy to pick up successful transformants. She also suggested that we read up on temperature-sensitive markers in bacteria and yeast to improve our project.
Dr. Dipshika Chakravortty, IISc Faculty
Dr. Utpal Nath, IISc Faculty
While Dr. Utpal Nath doesn’t generally work in this field, he gave us an outsider’s perspective on using cold tolerance as a marker for transformants. He said this could incur huge logistical costs in terms of having refrigerators constantly maintain these bacteria at low temperatures, as opposed to antibiotics which work at room temperature. Dr. Nath, like Dr. Varshney, put forth the point that laboratories aren’t contributing too much to the antibiotic resistance crisis - the main industries responsible being the health industry followed by the meat industry. According to Dr. Nath, one of the issues with the project was that the marker was not bimodal i.e. it would give us a gradient of cells which were growing instead of a binary answer. Antibiotics are preferred because of their flexibility, which wouldn’t be possible by using cold tolerance as a marker. To improve the project, he suggested that we have a graded scale of selection i.e. instead of selection at 4°C versus 37°C, we should have a stepwise increase with selection at 10°C wide slabs of temperature.
In addition to the above, we also had an extended discussion with Dr. Ramray Bhat (from the Dept. of Molecular Reproduction Development and Genetics, IISc) which was a deeply instructive, compellingly advocated, eye-opening conversation on a spectrum of topics ranging from synthetic biology, ethical regulations on research, the relation between society and science, the status and importance of undergraduate research, outreach programs, and some expert tips on making collaborations click. The complete text is available here.
Dr. Ramray Bhat, IISc Faculty
Industrial
As a part of our Integrated Human Practices exercise, we actively interacted and engaged with many stakeholders and professionals who were related to industries with a strong microbiological component. In each case, we sought to gain perspective on how growth/operations under suboptimal conditions have traditionally been a bottleneck, and also explored avenues to overcome the same using our cold-tolerant chassis in a contextually relevant and beneficial manner.
Shakesbierre Microbrewery
Our team representative had visited the Shakesbierre Microbrewery in Bangalore for a highly productive and insightful facility tour, which helped us develop a nuanced perspective. We learnt that the different flavors of beer are a product of specific yeast strains used for the fermentation of each. In particular, the secondary metabolites produced are responsible for the distinct flavors. The over-production of these secondary metabolites is usually controlled by suitable temperature adjustments including growth at suboptimal temperatures. With specific regard to our project, a potential application was suggested in the form of bacteria capable of stable growth at suboptimal temperatures which could allow for fermentation of novel flavors. However, this would be viable only if the increase in the chaperones and the change in temperature produces fewer/different metabolites which could change the flavour of the beer. Furthermore, the application of our chassis could also theoretically cut down on time but could very well increase production of secondary metabolites.
Biogas/Biofuel
Our team conducted a search for government-run biogas/biofuel setups within our state and came across a government scheme called National Biogas and Manure Management Programme (NBMMP), run by the Ministry of New and Renewable Energy (Biogas Technology Development Division). Taking this as a starting lead, we visited the state nodal center which was primarily responsible for the program implementation across the region - the Madhya Pradesh State Agro, Industries Development Corporation Ltd. Panchanan (operating from 3rd Floor, Malviya Nagar, Bhopal-462003).
Here, we found out that the established biogas plants were running on a small scale operational capacity (typically around 500 cubic square meters), and that they faced a shortage of sufficient biogas production during the winter season. To avoid this, they usually resorted to shutting down these plants over the winter. Further, we were directed to contact Mr Sunil Kumar Churay, who operated the largest industrial-level biogas setup within our state (located on Sukhi Sievanya, Vidisha Road, Bhopal, M.P.).
We spoke with his manager Mr. Rajesh Ambwani, who also voiced similar concerns over lower production rates during winters. Though they didn't possess any concrete data indicative of this issue, they conveyed that this was indeed a big concern on the industrial scale. Currently, they tackle this problem by running hot water across the body of the plant, using a fraction of biogas they produce, as fuel. In this regard, they did admit it to be a non-energy efficient approach, albeit a necessary one.
We then introduced them to our project and explained the idea we were working on - in particular, the ability of our system to potentially ensure that production rates at lower temperatures could be maintained. Though they were initially sceptical as to whether this could actually work out, towards the end, they were quite interested in our approach and heartily encouraged our efforts. In fact, they not only gave us direct inputs from their setups but also provided us with samples they regularly use as inoculum for the slurry in biogas plants. This contained many different types of strains of bacteria and other microbes that help in biogas production. On a positive note, they also indicated an active interest in employing our chassis on an industrial scale if it showed some promising laboratory results.
Dr. Vihang Ghalsasi
Our IHP team interacted with him via video conferencing on the 3rd of October, 2019, following which we received a variety of helpful insights on current industry practices and potential modifications to our ongoing project in order to make it more industrially relevant.
He initially explained that due to the issue of poor biomass yields at sub-optimal temperatures, current industry practices usually compensate for this by increasing the culture volume. While our system could help resolve this, predicting the consequences of a cold tolerant chassis on protein folding could be tricky - since the protein could either remain inside the inclusion bodies or get solubilized inside when expressed at a lower temperature. We were also told that while E. coli remains the most prevalent workhorse from an industrial point-of-view, it’s certainly not uncommon to employ insect cell lines for specific recombinant protein production tasks, for instance, antibody synthesis.
In order to troubleshoot, we were advised to look for specific proteins our recombinant chaperons could be preferentially adopting as substrates. On a related note, we were advised to browse through the Keio collection - an ensemble of all non-essential genes coding for non-essential proteins (as determined by knock-out studies) - and verifying whether Gro-EL/ES falls within this category. Following this, we could transform Gro-EL/ES mutant strains with our chaperons, and compare wild type with the recombinants thus generated. A particular helpful practical advice was to employ the vector pET-Duet, which has 2 different MCS sequences controlled by two different promoter-operator regions. This would enable the cloning of our construct in one of these sites by infusion PCR, while allowing for a check on recombinant protein production at the other MCS. The particular advantage herein would be a significant decrease in the plasmid load within the bacterial cell.
As an extension of our construct’s potential applications, it was suggested that we could explore cancer-specific biomarkers, express them in our chassis and look out for variations from the wild type expression systems. Similar to this, antibody production using our chassis could be another proof-of-concept since these are usually produced as small chains within the wild-type strains, which then accumulate inside inclusion bodies - a drawback that could potentially be overcome using our modified chassis. Furthermore, considering the possibility that our system could specifically enhance the production rates of soluble proteins, Dr. Ghalsasi advised us to test the same using certain soluble recombinant proteins (like enzymes) inherent to specific bacteria. A final suggestion was that we could possibly search for existing recombinant protein expressing systems and combine these with our construct, considering the time constraints we’re faced with.
On a concluding note, Dr. Ghalsasi exhorted us to continue working on our initiative, which he strongly feels has strong industrial relevance and broad-spectrum applicability.
