First Approach

Synthetic Biology has been described as a double-edged sword in multiple articles. On the one hand, it is viewed as this revolutionary piece of technology that allows us to manipulate biological organisms and processes and bring almost magical improvements to the quality of human life. On the other, the possibilities of misusing such a technology are endless. For every genome-edited baby, for every viral vaccine produced - there are hundreds of concerns raised in terms of the ethical and biosafety implications of doing so. And iGEM has always been a strong advocate of safe lab practices, identifying the potential risk groups in your project and ensuring minimal risk of misuse of your project.

At the very beginning of our cycle, we thoroughly went through the White List and the different classifications of organisms given by iGEM and the Australia Group, as well as the guidelines issued by the Department of Biotechnology, India^[1]. We strived to keep our project within the purview of the White List^[2] and guidelines of safe lab practices.

Our safety head attended the iGEM Risks and Safety workshop on June 9th. We had a lot to take away from the workshop. It began a survey on knowledge of biosafety and ethics and went into a description of all the safety and security policies of iGEM, like the release and containment of microorganisms. The hosts spoke of conducting responsible Human practices and ethical survey practices. They stressed upon the importance of informed consent, especially while talking to the vulnerable section of the population. They touched upon the legal considerations to undertake while shipping genetic material. They also emphasized the importance of anticipating and preparing for the worst outcome in any situation.

Ensuring a Safe and Ethical Production Method

At the time, our chosen therapeutic for Dengue was IgY. We found out that the primary means of acquiring IgY has been via the purification of egg yolk obtained from infecting chickens with dengue by injecting them with the virus. We also spoke with several experts who had worked with IgY before, and most of them had used purified egg yolk or a eukaryotic system. These methods have the multi-faceted problem of being unethical, having issues with batch standardization, and being polyclonal, which could lead to cross-reactivity. Determined to come up with a safe, easy-to-use, and affordable alternative, we chose the SHuffle B strain derived from the E. Coli B strain to be our chassis. E.coli, a Risk Group 1 bacterial organism, is safe to use and easily cultured. The SHuffle strain has its reductive pathways suppressed to make its cytoplasm predominantly oxidative and can produce virtually most complex proteins so long as the sequence exists - it proved to be an efficient, safe, ethical, modular, affordable, and an accessible alternative to using purified egg yolk. Additionally, it is cost-effective, retains protein expression over long-term culture, and does not have unwanted glycosylation as opposed to eukaryotic cell lines like CHO cells and eukaryotic systems like yeast. An important point to note about SHuffle is that since the modifications that suppress the reductive pathways are made to the genome, the risk of horizontal gene transfer in the unlikely but possible event of an accidental release is very minimal.

Lab Safety

At the beginning of our wet lab cycle back in March, all members of our team - both wet lab and non-wet lab underwent a 3-day long informal lab training course headed by our mentors Ashwin Uday and Gunwant Patil. We learnt several lab practices like parafilming, incubator use, hood use, pipette use, and working the different machines like PCR, Sonicator, Centrifuge, etc. Our mentors taught us all the important protocols like Gel electrophoresis, streaking and spreading, competent cell preparation, protein purification, SDS Page, alkaline Lysis, etc, and carefully monitored us as we performed it on our own. Most important of all, we learned about safety waste disposal and the different bins for plastic waste, liquid waste, culture disposal, the different areas designated for EtBr use, and SDS gel use. In the end, we had an informal quiz overseen by our PI, Prof Hotha, and were given little certificates and chocolates and were then considered certified to work in a lab.

Following the advice of our mentors, we have always performed all of our culture involving experiments in a BSL-2 hood in our Institute, and have strictly ensured that all our non-hood experiments were performed keeping all safety concerns in mind. All of our experiments were performed by our wetlab members wearing gloves, masks, protective gloves and coats at all times. We have separate designated spaces for EtBr use to run our agarose gels and for the SDS page to use AB mix. We also had separate bins for plastic waste disposal, liquid waste disposal and culture disposal. In addition, we also had separate disposal bins for all EtBr contaminated materials - liquids and solids. All of our experiments were performed under the supervision of at least one of our mentors.

Coming up with an Alternative for a Potential Non-White List Factor

While planning our assays for a Proof of Concept, we ran into a safety concern wall about using the Dengue virus. We could not use just the Envelope protein as planned earlier since we were told by Dr Milind Gore, previously from NIV, that the protein would not dimerise properly in the absence of the pRM-C structural protein, and a literature read also yielded the same conclusion. We also found out that that was the reason why most antibodies produced against vaccines with just the E protein lacked specificity against the E-dimer epitope.

We required the entire dengue viral protein minus the genetic material for our assays. Dengue virus (DENV) is classified as a Risk Group - 2 organism and is on the White List. However, since our target epitope contributed to the virulence of the organism, it fell outside the White List. We had concerns regarding containment and usage. We raised these concerns with the Safety team of iGEM and were advised to fill out a check-in form.

We sought to find a safe alternative for it and decided to work with the DENV-2 pRM-E VLP, whose cassette was deposited by Dr Stephen Harrison in Addgene. Dr Rahul Roy, a Dengue virologist from IISC, very graciously agreed to supply the same VLP produced in his lab to us and help us out with our experiments. He also offered the use of his lab space to safely run our experiments. We obtained permission from Dr Stephen Harrison and from the Material Transfer Department at Harvard, submitted a check-in and received the go-ahead from the iGEM Safety Team.

Shipping our Protein

We needed to ship our protein to IISc, Bangalore to run our VLP fusion assay to determine the neutralisation capacity of our protein. Before shipping, we did a thorough reading of the iGEM guidelines on shipping. We followed all the appropriate safety protocols in place by iGEM to ship it. We shipped 40 µL of our NeoFv kept in 2 cryovials filled with Dialysis buffer (the recipe can be found on the Experiments page) inside a ziplock bag. The bag was taped and sealed and placed between 10 kgs of dry ice. This was put in a thermocol box to prevent leakage which was again sealed and clearly labelled with the contents of the box, and the address of Dr Rahul Roy's lab at IISc, Bangalore. We shipped our protein via BlueDart. The shipment took fifteen hours to reach the destination. Our mentors were consulted regarding safe shipping methods.

Ethical Survey Strategy

Using the helpful tips about ethical surveys given out during the safety workshop, we came up with a survey to gauge the public's awareness of the disease, its pathology and the possible correlation of knowledge regarding dengue with the area they live in. We also sought to spread more awareness about the disease by including little tid-bits of information throughout the survey. Our survey was in a quiz format with answers and information given after every question. It does not ask for the survey takers' names to minimize accidental misuse of data. It only asks for their age, educational qualifications, and place of origin - all purely for analytical purposes.

Complying with the survey ethics laid out by our institute and iGEM, it does not ask for their medical history and lets the takers know that the data from the survey will not be misused and will only be used for representative purposes on our wiki. We sought the input of Dr Pooja Sancheti, an Assistant Professor at IISER, Pune, and modified our survey according to her suggestion of asking if our takers were from a tropical or non-tropical country to gauge the distribution of awareness.

Webinar on Safety and Ethics in Biotechnology and Research

In September, in a collaboration with the MIT_MAHE iGEM team, we invited Dr Vinod Jyothikumar, who is a consultant at DSS+, for a talk on Safety and Ethics in Biotechnology and Research. He touched upon the importance of:

• Biosecurity
• Lab Protocols
• Dual-use research of concern
• Supply chain and synthetic biology
• Ethics in cybersecurity
• Regulatory bodies, and regulatory aspects involved when it comes to building a project

The talk was followed by a question and answer session where he spoke of using CHO cells in research and their pros and cons.

Conclusion

We have thoroughly considered all aspects of our project and have concluded that while our project involves minimal risk, the possibility of a future misuse of our project or its components is possible. We have discussed the possibilities of entities using the FcRn binding peptide, which increases the half-life of the protein it is linked to in the body, to improve the half-life for certain proteins that can cause damage to your body. This can even be used as a bioweapon. We did look into switch-on and switch-off mechanisms for the binding of the peptide to the FcRn receptor to prevent this future possibility. However, we could not implement it due to a lack of time.