Team:IISER Bhopal/Human Practices/ihp
Integrated Human Practices
In the integrated human practices we aim to explain how interacting with stakeholders has shaped up our project. Contacting the doctors, research experts helped us shape our project by not only constructive criticism but also by questioning various aspects of the project which motivated us to seek answers and incorporate them in our project. Along with this they also provided with valuable suggestions which have been incorporated as and when required.
Key Highlights
This section discusses the summary of the findings and suggestions which influenced our project:
- As the currently available therapies for cancer have a plethora of side effects we aimed to create a solution with least to no side effects.
- We found Bifidobacterium longum to be a good candidate for solid tumors because it can colonise in hypoxic environments which can be present in solid tumors, along with that this bacteria is also a part of human gut microbiota, making it human friendly.
- The side effects of bacteria interfering with the system have been addressed with the help of tumor specificity and kill switch of bacteria, making it a targeted therapy.
- For proof of concept we were suggested to use E.Coli as the model organism which is also a facultative anaerobe and then after progress we can modify the systems for Bifidobacterium longum.
- We were only aiming to target the high lactate concentrations in the gene construct, but as suggested by research experts that we could also use hypoxic conditions of the tumor to induce gene expression, we decided to incorporate both hypoxia and lactate concentrations to increase tumor specificity for the bacteria.
- We assessed the opinions of the public with a ‘Cancer Awareness survey’ and found that there is a current need to make cancer services more affordable and we aim to create a solution which is economical and accessible to most of the people.
- We prepared a questionnaire for doctors to interview them about the treatment methods and approaches in real time, so as to get a clear vision for what to aim for. We were provided with insights about how radiotherapy is ineffective in hypoxic tumors and how our treatment as an adjuvant to radiotherapy can increase the success rate of destroying tumor cells, as our system can potentially sensitize these cells to the radiation exposure.
Our Interactions
Doctor Sanjeev Shukla
22nd March 2021
Getting Acquainted
Dr. Sanjeev Shukla is the Dean of Research and Development at IISER Bhopal (2018-present) and associate professor in the department of Biological sciences and his research work mainly focuses on cancer and the role of epigenetics in carcinogenesis.
Purpose
Through this meeting we wanted to understand about properties of cancer cells which we can use against them and so engineering the bacteria accordingly.
Discussion
- All cells express a basal level of lactate concentration. To construct a kill switch for our bacteria we need a lactate inducible promoter which is concentration specific.
- Bacteria and tumor growth kinetics are different (5 hours and 20 hours respectively). The bacteria will end up consuming nutrients of the media on co-culturing both bacteria and cancer cells, causing tumor cell death inevitably. We need to ensure that the activity of TRAIL and Smac are causing that activity.
- He also suggested that a hypoxia chamber for anaerobic bacteria culture can be created by using chemicals like CoCl2.
Integration
- For ensuring activity of TRAIL and Smac on cancer cells, we decided not to co-culture the bacteria and cancer cells. We planned to isolate the fusion protein and look for the cytotoxicity of the same on the cancer cells.
Doctor Partho Sarothy Ray
6th June 2021
Getting Acquainted
Dr. Partho Sarothi Ray is an associate professor in the department of Biological Sciences at IISER Kolkata. His research interest lies in RNA-binding protein and miRNA-mediated regulation of gene expression in inflammation and cancer, computational and experimental biology of gene expression networks, drug designing for cellular imaging and photo-dynamic therapy, molecular evolution.
Purpose
By meeting him we wanted a brief review of our project as well as wanted to know about the possible mechanisms for activation of the kill switch.
Discussion
- He made us realize that the effectiveness of our solution is limited mostly to hypoxic and solid tumors, since all tumors are not necessarily hypoxic and the bacteria (Bifidobacterium longum) grows in anaerobic conditions.
- He suggested doing the proof of concept in E.Coli as it is fit for almost any promoter and rbs.
- As every bacteria expressing proteins are not secreted outside the cells, he suggested going for a lysis approach in which contents of the bacteria are released outside into the tumor cells.
- He also gave us information about Hypoxia inducible promoters which can be found in Clostridium and Salmonella, as they will be effective in tailoring the gene construct as the bacteria senses the hypoxic conditions.
- Use of fusion peptide might not be a good idea, as it can impact folding of the proteins which might decrease their effectiveness.
- Tumor milieu is unstable in terms of extracellular matrix so biofilm formation won’t be taking place there.
- As all tumors are not accessible, we should go for intravenous administration of the bacteria instead of intratumoral, when it comes to drug designing.
Integration
- We decided to eliminate the plan of quorum sensing and incorporated an AND gate system for double proofing.
- We planned to use both Lactate inducible promoter and Hypoxia inducible promoter for the AND gate.
- For proof of concept we plan to use E.Coli DH5 alpha strain as it is a facultative anaerobe.
- This meeting formed the motivation for structural modelling of the native sequences and of fusion protein to ensure that folding remains unaffected.
Doctor Sandip K. Agarwal
8th June 2021
Getting Acquainted
Doctor Sandip Kumar Agarwal is an Assistant Professor in the department of Economic Sciences at IISER Bhopal and his research work is mostly oriented towards behavioral economics, agricultural economics and economic growth and development.
Purpose
Since he is a faculty of economics department we contacted him to get our survey vetted.
Discussion
- He briefly helped us to develop the survey with maximum adherence to the ethical and social guidelines which need to be followed when preparing an awareness survey.
Shrestha Rath
9th June 2021
Getting Acquainted
Shreshta Rath is a previous iGEM IISER Bhopal team member and is currently the after iGEM Regional Ambassadors Coordinator of Asia.
Purpose
We arranged a meeting with her to understand the safety considerations regarding our project.
Discussion
- She pointed out the problem of Horizontal gene transfer when it comes to working with bacterias.
- Risks associated with injecting the bacteria- they can elicit SEPSIS, and other immunogenic responses so we need to look for best possible ways to avoid such problems.
- She suggested looking for iGEM and outside iGEM projects pertaining to Bacteriotherapy to check for progressivity and novelty in our project.
Doctor Vikas Jain
17th June 2021 Getting acquainted
Getting Acquainted
Doctor Vikas Jain is an associate professor at IISER Bhopal (2015- present) in the Department of Biological Sciences and his work involves basic and applied research in bacteriophages and phage infection in Mycobacterium and thus developing phage therapy for tuberculosis.
Purpose
After validating an idea about the effectiveness of a fusion peptide of Smac and TRAIL we aimed to build a robust and effective fusion protein.
Discussion
- He suggested the use of serine and glycine linkers.
- He suggested using Histidine tag at the C terminus, after or before the linker.
- He helped us develop a cloning strategy in which 7 base spacers are necessary after the ribosome binding site can add 4 CCCC before ATG.
- Kill switch can be expressed as an operon system. He suggested inserting a Lambda repressor to balance the sensitivity of the kill switch and hence protecting the bacteria while traversing through the system to the tumor site.
Integration
- The kill switch and the fusion protein were constructed as guided by Doctor Vikas Jain.
Soumodeep Sarkar
19th June 2021
Getting Acquainted
Soumodeep Sarkar is iGEM Ambassador to Asia after iGEM and is the host of iGEM TV Show- SynBioFlix.
Purpose
We wanted to understand about safety policies of iGEM and hence we attended his workshop.
Discussion
- Team members’ safety should be primary when it comes to performing experimental work, followed by people the project we are doing for, animals and the environment.
- Certain organisms and parts are included in the white list which signifies organisms safe to use and allowed by the iGEM committee.
- When developing a therapeutic project one should consider safety of parts, safe wet lab work, and maximising use of organisms and parts on white list rather than those which are not on the list.
Doctor Sushma Agrawal
24th June 2021
Getting Acquainted
Dr. Sushma Agrawal is an associate professor in the Department of Radiotherapy, at Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India. She specializes in radiology and oncology. Her educational qualifications include M.B.B.S in General Medicine, M.D. and D.N.B in radiotherapy.
Purpose
By meeting with her we wanted a review of the project and as she is a doctor, she helped us understand the applicative aspect of our project.
Discussion
- Most of the access to the treatment comes down to the cost and effectiveness and associated side effects of it.
- ‘Solid tumors when grow big, their center becomes hypoxic. In clinical practice some tumors undergo surgery and then after the tumor is out then we give chemotherapy’.
- Side effects of the treatment depend on the modality of the treatment. ‘If we are using chemotherapy then there is myelosuppression, loss of hair, diarrhoea. And in targeted therapy it depends upon which targeted therapy we are using, but like basically,it is dermatitis, diarrhoea. Next type of therapy is immunotherapy which has a vast plethora of side effects, and depending upon the organ which is affected,and side effects can involve any of the organs and radiotherapy, side effects depend on where you are irradiating. If on abdomen then diarrhea, if on chest then lung toxicity.’
- Radiotherapy doesn’t work in hypoxic conditions, so we can use our approach to destroy hypoxic cells and increase radiosensitivity of the tumor.
An interview with a Cancer Survivor
4th July 2021
Getting Acquainted
The person had a history of Osteosarcoma which got detected in the first stage. The identity of the person has been kept anonymous and has been referred as ‘the person’ for the same.
Purpose
The sole purpose of the interview was to get an individual’s perspective who has gone through this ordeal and is now a major stakeholder in our project, and to understand how the general public will see the scope of our work. For the same purpose we also interviewed 4 more people who had a history of cancer or knew someone affected by it.
Discussion
- A pain for 10 days led to diagnosis of osteosarcoma of stage I in the right fibula with a CT scan.
- Six weeks of chemotherapy followed by amputation surgery as well as six months of chemotherapy were administered to this person in a span of 10 months costing about 5 lac rupees.
- The treatment affected almost all aspects of life and during the treatment the person encountered side effects like ulcers, vomiting, hair loss, tiredness, decreased appetite.
- The person, now a cancer survivor, believes in educating people about cancer and its treatments for making them mentally ready to undergo therapy in case they suffer from cancer, this will help reduce anxiety in the family and will make them come out stronger against the fight of cancer. Also, a psychologist in the department of cancer care might help people to face the ordeal.
- The person gave important advice - ‘If you feel any pain, get it checked, do not leave it’
Doctor Sanjeev Shukla
6th July 2021
Getting Acquainted
Dr. Sanjeev Shukla is the Dean of Research and Development at IISER Bhopal (2018-present) and associate professor in the department of Biological sciences and his research work mainly focuses on cancer and the role of epigenetics in carcinogenesis.
Purpose
The purpose of this meeting was to understand the modelling aspects which can be incorporated in our project.
Discussion
- Use of XIAP and Survivin is better when preparing the model for different IAPs as these IAPs are present in the solid tumors. To prepare a generalised model we need to do a stronger literature referencing how different IAPs are differentially expressed and regulated in various tumors.
- One possible drawback of our project can be that not all cancer cells express receptors for TRAIL, so effectiveness of our solution might decrease there. In such cases a combinatorial treatment might be better, like combining our solution with radiotherapy.
- Different types of cancers have different permeability and cells might develop resistance to our solution so we need to monitor the concentration of the amount being administered.
Doctor Nilesh Chandak
6th July 2021
Getting Acquainted
Dr. Nilesh Chandak is an Oncologist and is working in Chandak Cancer Care in Jalgaon, Maharashtra. He has his own youtube channel where he uploads videos of tumor surgery.
Purpose
The meeting was arranged with him to understand the application of our solution and to know more about how the currently available treatments are applied in practical life when it comes to cancer.
Discussion
- Cancers develop angiogenesis by VEGF pathways and that induces vascularisation. VEGF inhibitors are used to block these pathways which make the tumor partially hypoxic and then chemotherapy is administered. Currently both oral and intravenous injectables are administered for blocking these VEGF pathways.
- Chemotherapy is given when the size of the tumor is more than 1 centimeter. Cancers are known to exhibit genetic signatures which help determine whether chemotherapy should be given or not.
- The stage at which surgery is done will determine the percentage of recurrence and most of the solid tumors are treated with surgery. Like in the case of 1st stage of cancer if surgery is done then chances of recurrence is 10 percent.
- Drugs like Paclitaxel, Docitaxel are administered in case of chemotherapy so as to defunct the activities of mitotic spindle when tumor cells are undergoing mitosis. Drugs like Paclitaxel have irreversible side effects like neuropathies.
- Cancer cells need glucose for survival and it is metabolised to lactate and thus the increased concentration of lactate in the tumor microenvironment.
Doctor Abhijit De
13th July 2021
Getting Acquainted
Dr. Abhijit De is the Principle Investigator at Advanced Centre for Treatment Research & Education in Cancer at Tata Memorial Centre. His research interest is in Preclinical Molecular Imaging and Cancer. His lab involves miniaturized medical imaging equipment suitable for testing experiment concepts in animal models, which can be directly translated into imaging-guided clinical applications to serve both cancer diagnosis and personalized therapy.
Purpose
The main purpose of this meeting was to get a review and suggestions for our project and also look for possible help in modelling.
Discussion
- He was fine with the concept of our solution to be extended to most of the solid cancers and pointed out that oral cancer, breast cancer and glioblastoma are most hypoxic. Such tumors have a gradient of hypoxia and acidity in the tumor.
- Every stage has a different targeting strategy and late stage targeting is beneficial as conventional strategies might not have been sufficient to clear the cancer.
AIIM 2021
23 - 25 October 2021
From 23rd to 25th July AIl India iGEM Meet was organised with events organised by various teams from India. These included IISER Tirupati, IISER Pune, IISER Berhampur, IISER Thiruvananthapuram,our team IISER Bhopal and MIT Mahe. We had the opportunity to present our project in All India iGEM Meet 2021, in front of all the judges and the fellow iGEM teams from India. We received valuable feedback from the judges about the possible expenses which might come up during the commercialisation of our project and how we can work more on presenting the safety related issues of the project.
Doctor Mohit Jolly
17th August 2021
Getting Acquainted
Dr. Mohit Kumar Jolly was a Ph.D student in Bioengineering at Rice University and is currently working as an Associate Professor at the Centre for BioSystems Science and Engineering (BSSE) at IISc Bangalore.
Purpose
We wanted to understand about the possible modelling approaches and his guidance over the same.
Discussion
- As we demonstrated the references to him, he suggested checking whether the modelling studies are being done on normal cells or cancer cells, as they have different physiological features.
- He suggested going for MD simulations or Coarse grained analysis instead of kinetic models if we wanted to figure out internalization of proteins in the cells.
- We can also model hypoxic conditions for quantification of various factors induced by hypoxia.
- To determine saturation of ligands and proteins he suggested studying fractional killing of cancer cells.
- For approximating and quantifying data he suggested using values of GFP with CPP (Cell Penetrating Peptide).
Integration
- As suggested by him we decided to go for MD simulations and started contacting people who specialize in MD Simulations.
Doctor Rajesh K. Murarka
23rd August 2021
Getting Acquainted
Dr. Rajesh K. Murarka is an associate professor at the department of Chemistry at IISER Bhopal and specializes in Computational Biophysics and Chemistry. His research focuses on understanding conformational and structural dynamics of biological macromolecules and their interactions with the surrounding solvent environments.
Purpose
The purpose of meeting with him was to understand how to use MD simulations for understanding protein internalisation in the cells.
Discussion
- To start with we need to model the lipid bilayer system of tumor cells, as the properties of tumor and normal cells differ in physiology as well as in structural aspects.
- We need to understand whether the structure of the fusion peptide will change or not after cleavage, as these molecules are known to undergo structural changes when interacting and translocating across it.
- Driving force for protein internalisation can be either enthalpy or entropy, so one has to be clear over this as well as over the details of the energy barrier so as to understand affordability of the internalisation of the peptide in the cell.
- Atomistic model is a computationally expensive process and advanced sampling procedures need to be performed to accelerate and enhance the penetration and localisation of the peptide.
Integration
- We contacted Doctor Rajesh Murarka to start working over MD Simulations as a long term project to understand Smac internalisation dynamics.
Doctor Kavita Pal
24th August 2021
Getting Acquainted
Dr. Kavita Pal Bhayani is a faculty member at Biosetup Lifesciences and is also a scientist in Advanced Centre for Treatment, Research for Cancer - Tata Memorial Centre, Mumbai, India. She has vast experience in the field of nano-drug delivery, cancer biology and clinical studies.
Purpose
We had problems in figuring out methods to administer our drug, thus we wanted her insights over the same.
Discussion
- We need to create conditions for bacteria as much closer to the physiological conditions in vivo as possible in case of in vitro so as to check for the extent at which E.Coli can be used in anaerobic conditions as this organism is a model for our proof of concept. So, concentration levels of both lactate and hypoxia should be modified and changed so as to know more about the extent of bacteria’s optimal working.
- Immunofluorescence & immunoblotting & flow cytometry are a few techniques which can be used to visualize DNA breaks, caspase activation.
- When trying to develop a drug with bacteria one needs to see that how much bacteria will remain alive in case of Intravenous administration, it will depend on tumor location as well.
- Tumors have tumor specific markers which change according to size and type of tumor. So, intratumoral administration can be done or our solution can be merged with biopsy which uses tumor specific markers.
Doctor Rajesh K. Murarka
5th September 2021
Getting Acquainted
Dr. Rajesh K. Murarka is an associate professor at the department of Chemistry at IISER Bhopal and specializes in Computational Biophysics and Chemistry. His research focuses on understanding conformational and structural dynamics of biological macromolecules and their interactions with the surrounding solvent environments.
Purpose
To discuss over how to start working with MD Simulations
Discussion
- He suggested going for a coarse grained simulation because atomistic MD simulations will take a lot of time and hence not feasible. We are currently aided by one of his Ph.D students for the same.
- Immunofluorescence & immunoblotting & flow cytometry are a few techniques which can be used to visualize DNA breaks, caspase activation.
Doctor Maciej Maslyk
24th September 2021
Getting Acquainted
Dr. Maciej Maslyk is a professor in the department of Molecular Biology in the Institute of Biological Sciences at The John Paul II Catholic University, Lublin (KUL) in Poland. His research interests are into molecular biology, bioactive compounds, biotechnology & antimicrobial agents.
Purpose
As we incorporated most of the work of him and his colleagues in our project we had some doubts regarding the synthesis of fusion peptides and other experimental aspects.
Discussion
- Only the 7 amino acids of Smac/DIABLO were used on the N terminal part because this part is interacting with the BIR-3 domain and inhibiting XIAP.
- We earlier thought of incorporating whole Smac cds but that complicates the process of cleavage needed for exposition of the AVPI consensus. When using a full length protein, its activity will depend on the additional processes and its efficiency.
- His paper showed tumor regrowth when TRAIL alone was used as it acts in the feedback way.
- Tat protein is a good candidate for cell penetrating peptide.
Integration
- As he suggested we decided to go for a fusion protein rather than TRAIL and Smac separately and only the 7 amino acids of Smac were used which simplified its activity in the fusion peptide. For cell penetrating peptides his work inspired us to go for ones with better internalisation processes documented other than Tat, hence we went for Octa Arginine for assisting in internalization of Smac.
Doctor Sanjeev Shukla
28th September 2021
Getting Acquainted
Dr. Sanjeev Shukla is the Dean of Research and Development at IISER Bhopal (2018-present) and associate professor in the department of Biological sciences and his research work mainly focuses on cancer and the role of epigenetics in carcinogenesis.
Purpose
Meeting with him was arranged to understand about the experiments to be performed in the lab.
Discussion
- Ligation of gene construct in plasmid and transformation in bacteria can be used as primary culture. Secondary culture is prepared with 100 microliter of Luria Broth along with lactate and ampicillin in syringe and covering the syringe openings stops the air flow thus creating the hypoxia needed for Hypoxia inducible promoter to work.
- Using monoclonal antibodies to check for TRAIL-Smac expression.
- Using western blotting technique to check for cleavage of TRAIL and Smac and their sizes.
- We can use cell lysate to examine the cell extracts and check in which cell lines the TRAIL and Smac fusion peptide is cleaving. Weakness: The construct is unsuccessful in the cell line where caspase activation is not happening.
- Tumors have heterogeneous concentration of lactate and hypoxia, so it will not work in nearby cells, thus providing extra security to nearby cells.
Doctor Padma Devrajan
4th October 2021
Getting Acquainted
Doctor Padma Devrajan is the Coordinator of the World Bank Technical Education Quality Improvement Programme (TEQIP) at Institute of Chemical Technology, Mumbai. She is the professor in Pharmacy and former Head of Department of Pharmaceutical Sciences and Technology. Her work focuses on drug delivery systems for human and veterinary healthcare. (Linkedin)
Purpose
Meeting with her helped us understand about the formulation of the intravenous injection of Bifidobacteria.
Discussion
- The concern of bacteria being administered in veins was assured after the paper discussing the safety of intravenous administration was studied by us.
- Any soluble substance should be isotonic to the blood or plasma which has a molarity of about 0.3 M of solutes.
- Bifidobacterium longum will not be stable in the suspension medium so it needs to be freeze dried.
- To produce viable bacteria and stable products the freeze dried system is converted into powder and cryoprotectants like Trehalose are added to avoid stressing on bacteria during the freeze drying process.
- After freeze drying check for OD of bacteria as well as ensure homogenous distribution of the bacteria in the freeze-dried powder.
Richa Ahuja (Abiogenesis Clinpharm)
18th October 2021
Getting Acquainted
Richa Ahuja is the Senior Business Development Executive at Abiogenesis Clinpharm and is an experienced professional in the clinical research industry. Abiogenesis Clinpharm is a Clinical Research Organization (CRO) with expertise in providing Clinical trials of drugs, medical devices, nutraceuticals and other products.
Purpose
Meeting with her helped us to understand what needs to be done to start with human trials.
Discussion
- She suggested going to clinical trials once the trials and experiments with cell lines and then animal models are done. She helped us connect to Rodenta Bioserve for preclinical studies.
- The organisation provides trials from phase I to phase IV and she suggested going till phase II and then we can go for phase III after approval of DCGI.
- As per her suggestions, side effects of a drug can manifest within 24 hours, so the group under study needs to be observed for 2 to 24 hours to check for any side effects.
Doctor Kalle Anand Kumar (Rodenta Bioserve)
20th October 2021
Getting Acquainted
Doctor Kalle Anand Kumar is the Chief scientific officer and a founding member of Rodenta Bioserve. Rodenta Bioserve is a pre clinical research organisation which provides animal models and protocols for various research studies and assists pharma/graduate/postgraduate student research projects, private companies.
Purpose
We met him to gain insights about how to proceed with pre-clinical trials as they will form the basis for most of the invivo studies for various solid tumors.
Discussion
- He guided us for transition from in vitro studies to the in vivo studies and aided us with a protocol for a brief plan of action.
- He suggested the use of Rabbit (New Zealand White species) as a model organism to study the toxic effects of the fusion protein, if any.
- He helped us form a brief protocol for both the efficacy and toxicity studies of the fusion protein.