Overview
The 20th and 21st centuries have been the age of science and technology. The development of science has been influenced fundamentally by the needs and interests of society.
iGEM envisions a world in which what we develop goes beyond the lab, involving various stakeholders, people and every component of society that is affected. Here comes the importance of Integrated Human Practices (IHP). Our project is a new application to the pre-existing technology of Optogenetics and Cell counting. It is necessary for it to be well calibrated so that others can build upon it to develop its uses and advantages. Thus, it was important to take into account the suggestions and criticism from the people who are going to make use of it. Using this suggestions ,we altered our project making it more user-friendly.
The following is a brief overview of our Integrated Human Practices:
- Selection of a suitable method to measure cell ratio and to create a device suitable for researchers and industrial experts was our primary objective. We met them to discuss the feasibility, accuracy and the applicability of our project.
- One of our major concerns was the feasibility of the plausible applications of our project. Since our project was a foundation that could be adapted to be used in various fields, we talked with a wide variety of stakeholders from common people in the society, to industry experts
- We also discussed the aspect of the biosafety of our project with experts.
- Additionally, we prepared a survey to get inputs and the perspective of people involved primarily in research and industries.
Hardware
In order to sustain co-cultures successfully, we needed to measure the ratio of the bacterial population in the consortium at regular intervals. Generally, OD measurement is used to estimate the population of bacteria. In order to measure the population we needed equations to solve for the ratio of the population. We expected to get two independent equations by calculating the OD at two different wavelengths. Instead, we got a pair of dependent equations. This issue bothered us for weeks. After extensive research and brainstorming other ways to measure the population including impedance, viscosity, microfluidics and fluorescence tagging, we went on to look for labs having prior experience with these processes.
In order to choose the second method , we met with Dr. Aloke Kumar
Dr. Aloke Kumar
Department of Mechanical Engineering, IISc
Dr. Kumar’s lab works on understanding bacterial dynamics and its inter-relationship with hydrodynamics. They use microfluidics to investigate bacterial biofilms.
What we learned:
He was quite apprehensive about the impedance method, since it has not been extenively used before measuring the population. He mentioned that the viscosity method might not be reliable because it is extremely difficult to correct for errors caused by the incubator. He suggested the method of using zeta potential to determine the ratio of bacteria using the signal strength of different Fourier components of scattered light. If this is bi-modal (the zeta potential of the two are different such that the peaks are non-coincidental), we can determine the ratio of populations. He suggested testing the above principal using the zeta measuring device in the CeNSE (Nano-Sciences) department of IISc and cross verifying the populations by performing flow cytometry of the same samples. He mentioned that one of his students had used the zeta meter recently and guided us through the process of using the same. When we started the construction of the device, he asked us to use suitable voltage ranges and suggested the usage of electrodes that would not have antimicrobial properties unlike copper. He also mentioned that our project was quite unique and he hadn’t heard of any such thing before. He asked us to keep him updated on our progress and wished us all the best.
Thus, we proceeded with the impedance method. We constructed a basic prototype of the sensor. The device worked but the noise produced in the system due to vigorous shaking in a shaker was an issue. In order to resolve the problem, and to verify the reliability of the impedance measurement method in relation with our project, we decided to meet with Dr. Hardik Pandya.
Dr. Hardik Pandya
Department of Electronic Systems Engineering, IISc
Dr. Pandya's lab develops tools (sensors, microsensors, microfluidic devices), intended to solve biological problems using microtechnology and nanotechnology. His lab was then working on methods to measure the population of
Mycobacterium tuberculosis and mammalian cells.
What we learned:
After listening to the idea we proposed and our progress, he agreed that the impedance method seemed reasonable. However, what he pointed out was that the spacing of the electrodes that we were using was very big (in the range of millimetres). He suggested we use electrodes of a similar shape but with spacing in the order of micrometres instead. He himself got some of these fabricated from the CeNSE department of IISc to increase the sensitivity of our measurements. It also minimized the effect of change in volumetric geometry at a point.
Overall Project
At the initial stage when we were finalizing the idea, we wanted to troubleshoot the biological aspects of our project. As a managing director of a fast-developing START-UP and an ex-professor of Biology, Dr. Srinath Thiruneelakantan was the perfect person to visit. He knew what it takes for a project like ours to be adopted and developed upon by industries.
Dr. Srinath Thiruneelakantan
Founder-Managing Director, GenoPhe Pvt. Ltd.;
GenoPhe uses zebrafish as a model organism to validate disease hypotheses, faster in the screening library etc.
What we learned:
Biological Aspects
One of the major applications of our project was the production of rare metabolite. We wanted to prepare a list of useful rare metabolites which our system can produce. He suggested going through the available literature on transcriptome profiling. He advised us to go through changes in physical characteristics of Bacillus subtilis and E. coli due to high frequency or intensity of the laser, which could affect the OD measurement data. He asked us to find out if the effects of high intensity blue light were detrimental to the growth of bacteria and to make changes accordingly. Later, upon searching, we found that it induces stress in B. subtilis and so we decided to adjust the intensity accordingly. He also said continuous flashing would have a detrimental effect on the metabolic pathways of bacteria and shining it in pulses instead would be a better idea. We suggested the idea of a parallel processing bioreactor, wherein a bacterial consortium will produce an individual constituent in the desired ratio to produce one particular product. He advised us to look further into the downstream processing of the product formed.
Industrial and Hardware aspects
Dr. Srinath told us that even a small percentage of improvement is significant for industries because of the large-scale production involved. He also told us to clearly present the distinction and advantages between the existing hardware and ours.
In order to get a better industrial outlook, we decided to visit the Bangalore Bioinnovation Centre. The Bioinnovation Centre is a government created bio-technological hub where ideas are brought to reality. We wanted to discuss our project idea with scientific supervisors and executives of the companies that worked with bioreactors and fermentation. This session helped us understand the impact our project may have on industries and get some actual applications from companies.
Bangalore Bioinnovation Centre(BBC)
BBC is a start-up incubation centre. Here they have more than 300 companies are working or have worked in. The companies here work on a variety of biotechnological ideas from biocatalysis and tissue-engineering to various diagnostic kits.
What we learned:
We were going to build a small prototype of the hardware. They wanted us to consider the errors in measurement and other problems that become more significant when measurements are scaled up to implement this on an industrial scale. For measuring the growth using the OD method they wanted us to create a standard curve every time we changed the constituents in the bioreactor. They wanted us to analyse the variations in measurement with the pH, salt concentration and other parameters. They also wanted us to look into the downstream processing of the protein produced through parallel processing.
Applications suggested by them
- Production of enzymes of different required reactivities(in the bioreactor) and the control of their activity would be made much easier using our setup.
- Probiotics producing bacteria can be co-cultivated easily using our optogenetics-based hardware as probiotic proportions can be changed easily.
- Saravanan Jothi, the scientific manager of Iosynth, said that the optogenetic method which we presented had the potential to replace various other methods they are currently using, such as heat-sensitive promoters, growing other microorganisms to control the growth of bacteria in the bioreactors etc.
Modelling
As our team did not have any prior experience in the modelling a co-culture of 2 bacterial populations which was closely related to that in Bioreactors, we decided to approach Dr. Jayant.
Dr. Jayant Modak
Department of Chemical Engineering, IISc
Dr. Jayant is a deputy director of IISc and his lab works on modelling of fermentation processes, optimization and control of bioreactors.
Why we met him:
To help us in modelling the co-culture system. He had vast experience in the modelling and optimization of Bioreactors.
What we learned:
He provided us with some valuable advice for modelling the system. We were asked to start with modelling the system with a basic growth equation for a single bacteria of the form (he asked us to initially assume independent growth and then add correction terms for the bacteria as necessary)
He also asked us to include the rate of change of concentration of substrate depending on the growth rate, death rate and present number of bacteria in the culture. He finally wished us the best for our project and asked us to keep him updated.
Survey-SynShine: Feedback & Suggestions
We prepared a survey in order to get inputs from people involved in R&D and academia whom we cannot reach directly for interviews
Survey: IHP
Aims of the survey
- To gauge the usefulness and limitations of our project to society.
- To estimate the probability that industries would implement our project.
- To ask for suggestions for improvement that can be incorporated to make our project better.
Important Results from our survey relevant to Integrated Human Practices
- In order to rate the relevance of our project to society ,people were asked to evaluate, the usefulness of our project to the present world. It was rated 4.3/5
- Question was asked to analyse the feasiblity of industries to implement our project. Our project was rated 3.8/5. 1 being least likely; 5 being most likely to be implemented
- Question was asked to scale the complexity of operating hardware for an average factory worker. It was rated as 3.267. 1 being very simple ; 5 being very complicated
- Question was asked to determine the major limitations of our project with some common limitations as option. 40% of people considered the high cost to benefit ratio, high running cost and expensive maintenance as the major limitation of our project.
- Question was asked in order to analyse how much industries are ready to invest on new technology. About 50% of the respondents thought that the industries wouldn't be spending more than the cost of the existing technology.
- Question was asked to know the major advantage of our project from people involved in R&D. Helping in drug discovery was considered the major advantage of our project, followed by the synthesis of rare metabolites and the improving of efficiency of Bioreactors by enabling parallel processing.
- Overall, our project was rated 4.13 on a scale of 5.
Suggestions provided by respondents for improving our project:
- Co-culturing pathogenic bacteria to study their interactions.
- Decreasing cost and increasing the procedural efficiency.
- Applications in recombinant DNA technology
1-0% ; 2-0% ; 3-6.7% ; 4-53.3% ;5-40%
1-0% ; 2-0% ; 3-40% ; 4-40% ;5-20%
1-6.7% ; 2-13.3% ; 3-40% ; 4-26.7% ;5-13.3%
a-53.3% ; b-26.7% ; c-53.3% ; d-66.7%
