Validating the hypothesis

Dr. A. Senthil Kumar

Professor & Division Chair, Environmental & Analytical Chemistry Division

The CO₂ equilibrium is always established in nature. With the increase in CO₂ emissions with industrialisation and other anthropogenic actions, the overall CO₂ concentration in the atmosphere has increased. Considering the equilibrium that nature is trying to establish as given below:

From the above equation, it is clear that with increasing CO2 in the reactants, with respect to Le Chateliers principle, the forward reaction is favoured and there is more dissolved CO​2​. This establishment of equilibrium is what causes the increased CO2 level in the ocean and thereby leading to ocean acidification. Initially we had decided to use a system that facilitates an influx of carbonate/bicarbonate ions. Thankfully, our discussions with Dr.A.Senthil Kumar after almost 2 months of working brought in light the fact that by using a system that removes products from the above reaction, will tend the reaction to move in the forward direction and that would mean we were doing the reverse of what we intended i.e., we were going to reduce the pH by dissolving more CO₂.

This was a major contribution after which we changed our system completely.

V. S. Saravanan

Asst Professor, ​ Department of Microbiology ,Mūvattupula, India

Since our system leads to a high concentration of H⁺ ions in the cell, we were doubtful of its fate inside the organism. Dr.Saravanan very enthusiastically explained to us that H⁺ions are majorly used for energy generation,and once homeostasis is established the excess H⁺ ions are flushed out of the system.

Choice of organism

Our organism of choice should certainly be saline friendly, be able to survive varied pH and also accommodate the system that we have in place.Early in our project, we got on a Skype call with iGEM Marburgh who are currently working with ​Vibrio natriegens. ​ Our discussions with the team further interested us to use ​V. natriegens as a model organism because of its less doubling time and its necessity for 2% NaCl for its growth (thalophilic). This was not put into action because of the difficulties in protocols. Our experiments were contained to ​E.coli DH5 Alpha. With due course of time, multiple discussions with professors gave us a clearer picture on how to find our desired organism for future prospects.

Jabez Osborne

Professor Jabez Osborne suggested the following:

• Analysis of the stressful conditions that the organism will face in the proposed environment
• Suggested different protocols to find our desired organism
• Emphasised on the need for using native organisms instead thereby enabling easier acceptance to the natural environment
• Also suggested the use of Macro algae

Prof. M.A. Vijayalakshmi

The Director of Molecular Interactions and Separations Technology Labs at LIM Tech. S, UTC, France

The Center for Bioseparation Technology (CBST) was created at VIT with Prof. M. A. Vijayalakshmi under the "High priority Research Area" funded by Department of Science and Technology (DST), Government of India. She was identified by the Indian Government to come back from France after more than 30 years of service at Universite de Technologie Compiegne (UTC), France, to steer this prestigious center.

Firstly, the abundance of knowledge that she had to share with us and her loving and humble nature inspired us. With decades of experience in the field, her insights on our project was a great contribution to view our idea in the bigger picture.She emphasised on the fact that the environment in even the different oceans were going to be different and that we have to work with specific areas to be able to get the native organism that we can possibly manipulate.

Dr. Sridharan T. B.

Professor of Department of Biotechnology VIT, Vellore

He suggested the incorporation of our system in seaweeds and phytoplankton to achieve faster growth. He also mentioned the prevalence of organisms is zone specific.

Joseph Selvin

Department of Microbiology, Pondicherry University

With respect to his work, Dr. Joseph Selvin explained his observation on the effect of temperature on the symbionts of coral reefs. In his first year of observation, he saw a decline in the corals and its associated symbionts. But over the next two years of his observation, he saw the revival of the symbionts suggesting that they were evolving according to the environmental changes. The result of his work suggests that similarly it is possible that over a period of time,an evolution might occur in organisms native to the ocean enabling them to resist the change in oceanic pH. He also suggested that we keep in mind that the metabolic pathways of the organism in concern should be sustainable in alkaline environment as well.

Dr. Punyasloke Bhadury

Associate Professor, Biological Sciences, IISER Kolkata

Dr. Punyasloke Bhadury is a scientist who predominantly works on using biological systems to tackle ocean acidification. We reached out to him as his area of interest was in par with our problem statement. It was an amazing opportunity for us to speak to someone with such expertise. He gave us a deeper insight into our model. He was enthusiastic about the project and was in favour of using indigenous strains as they have a good doubling time and carbon dioxide capture capacity.

He suggested that we also need to look into how the engineered strain will behave at different parts of the oceans, like at the coasts and further into the ocean. He also proposed a parallel mechanism that can work complementary to our existing system, of using Carbonic Anhydrase. He suggested that express enzymes that are inherent to micro algae that can increase the efficiency of the whole construct.

Choosing the suitable system

Jabez Osborne

Assistant professor(senior) - Dept of Biosciences VIT, Vellore

In our initial proposal, we had planned to use PhoBox promoter for sensing the external pH. This works using a periplasmic protein PhoR which phosphorylates PhoB that acts as the activator of the PhoBox promoter leading to translation and expression of the necessary proteins to control the pH.

In our discussion with Dr. Jabez , he asked us to check the activity range of the different proteins and promoter system involved in our project at varying factors like pH and temperature. When we did check for it, we found that the promoter is active at pH below 7. But the oceanic pH is around 8.So the PhoBox promoter wouldn’t work at that pH. This led to the modification of the idea into its current form.

Safety

Everette Jacob Remington N

Associate Professor of School of Bio Sciences and Technology & Member of Institutional Biosafety Committee and Stem cell Research Committee

His insights on safety regulations related to GMOs gave us a clearer picture on why regulation of our organism’s mechanism is of high importance. His suggestions led us to think of incorporating our RecA-LexA system.

Mathematical Modelling

Mrudula Pulimi

Chemical Engineer by profession, Ph.D.

An excellent advisor who has worked on mathematical modelling and Nanotechnology based heavy metal remediation solutions and specialised in Simulink, MATLAB​.​She played a vital role in our mathematical modelling by helping us understand the concepts in a simple manner. We had complex ideas and we were unable to run the program on MATLAB. She helped us to simplify it and gave the idea to incorporate our H⁺ ions in the equations itself.

Future Prospects

Sudandira Doss C

Associate Professor-Department of Biotechnology VIT, Vellore

In our discussion, he proposed that we perform scale up studies and understand how much of inoculum to release into our desired environment.

David Ishee - a Virtual Conference

As part of Integrated Human Practices, we reached out to Mr. David Ishee, a well-known biohacker. Mr. Ishee is a passionate dog breeder who was stumped with the idea that there wasn’t a way to tackle an inherited disease in Dalmatians called hyperuricemia. Hence, he took it upon himself to find a way using genetic engineering as a tool and as inexpensively as possible. He started out his own venture to face this issue and has hence inspired several to use synthetic biology as a tool to face many problems.

We had a virtual conference with Mr. Ishee over Skype and he took us through his initial research with Dalmatians and the difficulties he faced with respect to rustic genetic engineering in his home-lab. He talked us through the CRISPR technology and how unique and easy it was use it for modifying each strain. He also suggested how our organism of choice interacts withotherorganisms in the native ecosystem and how it might affect our project.

Tamanna Sharma

Founder & Director, Earthling First Private Limited

Miss Tamanna Sharma is a Journalist by profession but was inspired to start her venture when she recognized that the waste management was a serious issue that needs to be tackled. This laid the foundation for Earthling First, Miss Tamanna’s own venture to a better and a sustainable future. Earthling First has collaborated with various initiatives by the Government of India.

Our talk with Miss Tamanna Sharma was very insightful and enriching. Even as she was not an expert in the field of genetic engineering or its related fields, her experience working with nature and the people affected by the disasters caused by man, made her a very resourceful person.
Even as Miss Sharma was against the concept of genetic engineering as it was a lot against nature, she conceded that it could be beneficial if done safely and keeping in mind that the natural systems are not to be tampered with to a large extent or in the future. She was favourable to our idea of using synthetic biology to tackle Ocean Acidification as the phenomenon was a man-made disaster and it was up to us humans to find a solution too.

She firmly said before any actual release of any of our engineered organism into the natural oceanic waters, we need to simulate how our organism would respond to the such environments and this should be done by taking oceanic waters were taken as a sample for simulation. Moreover, she was cautioned us about how our engineered organism could potentially affect the natural balance of the marine ecosystem and that too needs to be monitored and simulated carefully before any release into the natural waters. It really is true that experience is the best teacher!