Team:IISER-Pune-India/Human Practices
Human Practices
In the very beginning of our iGEM journey, when we were brainstorming for a project idea, we decided to talk to young children. Our motivation for this was that if a child knew about an issue then it surely must be very prominent and a widely talked of problem. We approached children of grade 7 from underprivileged backgrounds who were part of IISER’s social initiatives program, DISHA. We asked them the question- What would they like to see changed or improved in their surroundings? Their answers were a varied bunch, from better schooling to reduced pollution. However, one answer remained consistent. More than two-thirds of the kids complained about the dismal state of the rivers or lakes that formed an important portion of their surroundings. They noticed that because of the garbage pileup and industrial outflow, the river was teeming with undesirable weeds, and fauna like fish and birds were decreasing by the day. Because of the pitiable state of these rivers and lakes, there were increased incidences of water-borne diseases and ailments in their communities.
We were always reminded of these kids whenever we thought about a potential application of our project. This is one of the main reason that we wanted to evolve a bioremediation system that helps clean water. Serving as a proof of concept, it could do much more. It would help these kids get access to clean water, thus reducing instances of water-borne diseases and ailments. Now, they too could take clean water for granted and work towards a better future, for them and their families.
During the development of our project, we interacted with many experts and stakeholders in the field. This helped us in refining our project to a great extent. We tried integrating ideas and advice of different experts into our project. The applied design of our project is completely designed based on our conversation with Mr Robert Pintabona, the Program Manager for Plants, North East Ohio Regional Sewer District (NEORSD), Cleveland. Here are the details of our Integrated Human Practices:
To understand the intricacies of taking a project from lab to industrial scale, we had a SKYPE conference on the 25th of August, 2019 with Dr. Vanishree Srinivas, Ph.D. Microbiology and former CEO of PRONET Informatix.
We started off by describing various aspects of our project to Dr. Srinivas. When presented the idea of increasing the mutation rates in E.coli through the incorporation of MutD5 in the system, she posed the following questions - How stable (resistant to mutations) is the bacterial genome considered? Would manipulating one DNA proofreading pathway actually result in a significant increase in mutation rate without an alternative pathway kicking in? As a reply to these questions, we provided her with literature which showed increase in mutation rates upon the introduction of MutD5 into the system and the figures of fold-increase provided in them [1].
The discussion continued to how our system maybe used as an effective tool for directed evolution. Here, Dr. Srinivas brought to our notice the low likelihood of achieving the necessary phenotype through mutagenesis as a biological system is indeed very ‘SMART’ and producing mutant versions of genes more not necessarily show a phenotype due to alternate pathways stepping in to ensure normalcy.
After describing the project, we asked her to comment on whether such a tool could be brought into the market and if so, what are the necessary steps to achieve that goal. Dr. Srinivas’ reply was that answering the following questions are clearly presenting the corresponding data to investors was the most crucial step. The questions include - What is the possible range of mutation rate achieved by the system? In what aspects would your system be better as compared to current techniques and its efficacy? How efficiently does it work across bacterial strains other than E.coli. She urged us to look up the various patents on different techniques used for directed evolution as a preliminary step. She provided some examples, such as the current patents held on mutagenesis through DNA shuffling and the technique using biopanning of virus vectors [2,3].
With respect to the lead bioremediation system, she told us of the importance of having a bioremediating bacteria instead of chemical methods to clear heavy metal from water. Points in its favour were high growth rate of bacteria and hence not much material to secure, efficiency of absorption/removal, ease of removal of bacteria from the water (if no toxic secondary metabolites are produced).
She asked us to keep the following points in mind while presenting the system - Comparison between natural bioremediating strains and the bioremediating strains we hope to evolve based on - What is the amount of lead absorbed or adsorbed by them? What is the amount of lead it is able to remove from the medium and the corresponding efficiency? Upto what level of lead in the environment can the bioremediating strains survive in?
Dr. Srinivas concluded the discussion by stressing upon how the most primary thing investors look for are sound well-documented results and only upon producing those will we be able to convince them to convert our idea into a marketable tool.
References:
[1] Selifonova, O., Valle, F., & Schellenberger, V. (2001). Rapid evolution of novel traits in microorganisms. Appl. Environ. Microbiol., 67(8), 3645-3649.
[2] Lutz, S., & Patrick, W. M. (2004). Novel methods for directed evolution of enzymes: quality, not quantity. Current opinion in biotechnology, 15(4), 291-297.
[3] Xiao, Xiao, and Lin Yang. "Directed evolution and in vivo panning of virus vectors." U.S. Patent No. 8,632,764. 21 Jan. 2014.
The Team had an online conference with Mr Robert Pintabona, the Program Manager for Plants, North East Ohio Regional Sewer District (NEORSD), Cleveland to know more about sewage treatment plants and to discuss the construction and feasibility of a device that uses engineered bacteria to sense and remediate lead ions from water. We started off by discussing the basic plan of sewage treatment plants and how they function.
The average inflow of sewage in a plant is about 35 million litres per day, for a plant serving a population of 100,000 people. The construction of a sewage treatment plant demands that the collection should be at the lowest part of the city, and the treated water is then pumped up and out. Thus, the hydraulic profile of the sewage treatment plant puts certain constraints on how our device can be used.
A sewage treatment plant has multiple steps of treatment for wastewater. The quantities used to determine the degree of pollution are Biological Oxygen Demand (which gives a readout of the organic material in water) and ppm levels of different elements. The basic plan of treatment plant involves the following processes-
1. Sedimentation of heavy waste using physical treatment methods
2. Bacterial treatment (both anaerobic and aerobic) to reduce the organic matter and the BOD of water
3. Chemical treatment to precipitate unwanted compounds and disinfection
Using current methods, lead removal would be classified as a part of the chemical treatment. With our engineered bacteria, this treatment becomes biological. However, according to Mr Pintabona, the treatment would not be as simple as inoculating the water with our bacteria. As lead is toxic to the bacteria as well, a separate compartment would be required where bacteria containing lead are continuously removed and fresh bacteria are added. The bacteria has to be grown in the wastewater for a time sufficient to sequester lead which can slow down the flow rate of the sewage treatment plant. Moreover, the lead content is diluted in such a huge volume which would decrease the efficiency of our bacteria. Thus, using lead sequestering bacteria in a sewage treatment plant might not be very efficient.
The major source of lead in any water body would be industries surrounding it. Mr Pintabona has suggested that industries might be better suited for an alternative method of lead sequestration. A similar contraption would be suitable and the efficiency is expected to be higher as lead concentration and the volume of water to be treated would be more. The bacteria is also a cheaper alternative to the current chemical methods being used as there is no byproduct, and removal of bacteria is fairly simple as compared to removal of chemical components. This method is suitable for reducing lead pollution from point sources, but can not be used for more spread out sources of pollution such as runoff from agriculture.
Mr Pintabona also raised the question about what happens to bacteria that have been exhausted with lead. Currently, we have no plan of extracting lead from the bacteria. Thus, for disposal, the bacteria will be killed and they will be disposed of in a special landfill that has the capacity for heavy metals.
Based on input from Mr Pintabona, we have tried to design a device that could be used in industries that can help reduce the release of lead. However, our device is not compatible with reducing the existing amount of lead in rivers and other water bodies because of technical limitations.
Since this is a mutagenetic project, we have taken a lot of care to ensure the safety of the environment. Check out our techniques for Safety.