Safety of OviCloak

• Choice of commensal-

Millions of commensals colonise the upper female reproductive tract. In our project, we plan to engineer a commensal, i.e. lactobacillus, to avoid harmful effects on the human body. We chose our commensal, which would primarily reside in the desired region; in our case, it’s the fallopian tube. The points which favour commensal’s inhabitance in the fallopian tube are- 

1. pH variation

The trend/variation of the pH in the female reproductive tract is that the lowest pH is in the vagina (4.42), increasing with the pH of nearly 7.94 in the fallopian tube, and then the pH of the ovarian fluid is maintained at 8.5. Our commensal (lactobacillus) mostly stays in the fallopian tube, i.e. at a pH of nearly 7.84. And as we know, the commensals reside in a particular pH.

[a pic of the female reproductive tract, divided in horizontally(3) and each showing pH of that area]

1. Direction of ciliary moment

The luminal cells in the fallopian tube have cilia on their surface, and its movement is from the ovary to the uterus. And as our commensal bacteria lactobacillus in inmotile, it will move mostly in the direction of ciliary movement. Hence, decreasing the possibility of it going towards the ovary.

• Ovastacin- a native protein

We are engineering our commensal bacteria to produce an Astacin-like metalloendopeptidase named ovastacin. Ovastacin is a cortical protease produced in humans for the post-fertilisation cleavage of ZP2. Hence there will be less possibility for this protein to cause any problem in the human system. 

• Horizontal gene transfer

One of the associated concerns with our proposed contraceptive OviCloak is horizontal gene transfer. After the delivery of the bacteria to the site of fertilisation, i.e. the ampulla region of the fallopian tube, there are chances for the transfer of artificially constructed plasmid to the natural bacteria present in the fallopian tube. Considering this, we propose integrating our desired genetic circuit and cassettes the genome of our bacteria lactobacillus with the help of cloning. This process is known as genome integration. This method will lessen the possibility of horizontal gene transfer as genome size is quite long, which will take to get entirely transferred in the other bacteria. But for the horizontal gene transfer, the pilus is not stable for a long time. Hence, there happens to be very little probability for the genome to transfer.

• Biosafety kill switch (Biocontamination control)

We have engineered our bacteria with a kill switch to avoid escaping them from the reproductive tract and avoid biocontamination. For more information about biosafety kill switch.

• Dual use-

1. Due to the involvement/enforcement of other people, women might not be able to use xylose for the induction of kill switch due to the involvement/enforcement. Hence, women using this contraceptive might not be able to induce the kill switch for reversibility. 
2. On the other hand, someone can use ovastacin and inject it into the ovary, which will cleave the ZP2 of the oocytes forming in the ovary and making the women sterile completely.

Lab Safety

Safety and Security plays a vital role in performing responsible research and hence, our team strived to practice safe and secure lab practices.

We initiated our safety practices with risk assessment to find the risk involved in doing the intended experiments and to strategize how to manage them.This was followed by the filling of the preliminary safety form

We underwent a safety training program facilitated by IISER Tirupati during the initial days of research activity in our lab.This helped us understand the potential sources of risk and the management/disposal of biohazard, chemicals and physical hazards.

During our project experimentation, we worked mainly in the undergraduate lab of IISER Tirupati. This is a BSL-1 Lab with two Class-1 Biosafety cabinets. We strictly adhered to the general safety guidelines and in particular the biology department safety guidelines.iGEM safety rules and policies were also adhered while doing research activity in our lab.While working in the lab we used appropriate personal protective equipment like lab coats, long pants, nitrile gloves and shoes.

Waste disposal

We segregate the waste produced in the lab as Sharps, biological solid/plastic waste, biological liquid material waste, and other solid waste.Sharps were collected in a puncture proof sharp container. Biological solid/plastic waste are collected in the red-biohazard bag and autoclaved for decontamination and  picked by the specified agency of the institute for incineration.While the liquid biological waste was contained in a container and treated with 10% bleach and kept overnight before discarding.Other solid wastes are collected and discarded off with other waste.

Waste containing hazardous chemicals like Ethidium bromide containing agarose gel, SDS gels were discarded separately and picked by the specified agency of the institute for incineration.Organic solvents are disposed separately from other liquid solvents, so as the plastic wares contaminated with organic solvents.

Organisms, parts and activities

During our research activities, we usedEscherichia coli DH5- Alpha, B.subtilis 168, Saccharomyces cerevisiae, strains which were risk group 1 organisms.

Moreover, All the parts and activities were in the white list

Lab safety under the context of COVID-19

To avoid contributing to the spread of COVID-19, we worked considering our local conditions and followed local and national public health guidelines. We were quarantined after our return from local states to the institute as per institute guidelines. Thereafter, we maintained social distancing and responsible interaction during our lab work including wearing masks and frequent sanitizing. We made sure that we are strictly adhering to the COVID-19 protocols followed in the institute.

Safety and Security - Human Practices

Safety and Security are crucial when it comes to human practices as it involves interaction with people during this pandemic scenario.

Safety and Security

We thought of this from the planning of our activities. We decided:

• To engage people virtually

All the public engagement and communication events were held online. We thought of utilising social media as a platform for outreach and education.

• Practice Social distancing during in-person interviews

When the Covid cases went down in number, we thought of meeting experts in-person. But we strictly adhered to COVID guidelines such as wearing masks, sanitising, and social distancing. 

• Engage with family-members

We planned to do In-depth interviews to reflect the needs and necessity of people. Under the Covid scenario, this was not possible. Hence we thought to start our IDI from our homes, with our family members.

Responsible Research

For a responsible conduction of our Human practices, we made sure the following :

• Follow Social research ethics and it’s regulations

We observed UNESCO’s Code of conduct for Social Research, while carrying out surveys and IDIs. We also got sanction from the ethics committee of our institution for carrying out our surveys.

• Collect Informed Consent

We made sure to collect written informed consent from individuals participating in our surveys and In-depth interviews. We made sure that the informed consent contains:  

1. Information about the data we are collecting and the purpose and most importantly we informed them that the participation is voluntary and they have the right to discontinue their participation at any point of the data collection process without any consequences.
2. A checklist to confirm 
1. They have had the chance to ask any questions regarding the project.
2. They understand that their participation is voluntary and that they could terminate their contribution at any time without consequences.
3. They agree to participate in the study and (if applicable) they agree to be recorded.

We also checked with our institute on the format of informed consent and got approved.

• Anonymising data

We assured our participants of the survey that the data collected will be anonymous.While, We collected written permission for publishing the data collected from experts during in-depth interviews for integrated human practices.We also paid attention whether our any of expert is an EU Citizen, so that we could follow General Data Protection Regulation (GDPR).

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This is extra:

General safety protocol

http://www.iisertirupati.ac.in/institute/safety.php

• Safety training by iiser tirupati
• Wear
• Safety levels of our lab

• Teams must think carefully about in-person interactions, whether between team members or with others in the community. To avoid contributing to the spread of COVID-19, consider your local conditions and always follow local and national public health guidelines.

Safety assessment - safety form

Safety Guidelines from institute and iGEM safety rules and regulations.