Team:IISER-Pune-India/Safety

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Safety

With great powers come great responsibilities

General Laboratory Safety

The iGEM Team IISER-Pune is in full compliance with the safety and security rules of the iGEM competition.

The team was included in the laboratories of Dr. Chaitanya Athale (IISER-Pune) and G1-Undergraduate lab (IISER-Pune), which are equipped to be biosafety level 1 (BSL-1) facilities. This level is the lowest biosafety safety level and was sufficient for all our experiments. Before we started our work in the lab, all team members had to pass different safety tests and receive training during an introductory wet lab safety week. Those tests included an introduction in the laboratory machinery (i.e. thermocycler, centrifuges, gel chamber), and also covered the use of chemicals (i.e. concentrated acids, phenol, and other reagents). Moreover, we had participated in the mandatory institute’s security introductions, including explanation of reanimation equipment, first aid and escape routes. Additionally, we had to pass a written examination on biosafety before commencing any laboratory work. This test was taken by Dr. Chaitanya Athale to make sure that we were aware of all the risks and safety protocols. At all times, a supervisor or instructor was present when work was conducted in the laboratory.

Project Safety

The chassis organism will be transfected with plasmid with chloramphenicol resistance marker. The plasmid insert will have systems that would allow for high mutation rate and lead remediation. This can allow the bacteria to thrive in the presence of chloramphenicol and low concentrations of lead. This can also cause in gain of function (GOF) in the bacteria or easy adaptation to multiple stresses. This in rare situations can lead to multidrug resistance and other hazardous functional mutations.

Thus, there are multiple safety methods that can be taken to reduce all biosafety risks. The methods can be implemented to either kill the bacteria or to cure it of the plasmid. The methods will be as follows:-

Ts ori plasmid sacB sacR system

A temperature sensitive ori-site can be introduced in the plasmid. The ori does not allow for replication of the plasmid at high temperatures and without the plasmid, the bacteria will be unable to survive in the presence of antibiotic. This will allow for killing all bacteria transfected with the plasmid by simply heating to/above a certain temperature or by cooling beyond a certain point [1].

It is seen that in the absence of antibiotic, bacteria readily loses plasmid with antibiotic marker due to the conferred cost of preserving the plasmidṣ. The plasmid can have an extra insert consisting of the sacB-sacR gene under its native promoter. This in presence of sucrose, produce levan which is toxic to gram negative bacteria [2]. Thus, transfected bacteria can be grown in the absence of antibiotic to cure plasmid and bacteria with plasmid left after this can be killed with sucrose (negative selection [3]) and bacterial cells without plasmid can be retrieved.

References

[1] Phillips GJ.(1995). New Cloning Vectors with Temperature-Sensitive Replication, Plasmid. 1999 Jan;41(1):78-81.

[2] Liu Y, Miao J, Traore S, Kong D, Liu Y, Zhang X, Nimchuk ZL, Liu Z, Zhao B. 2016. SacB-SacR gene cassette as the negative selection marker to suppress Agrobacterium overgrowth in Agrobacterium-mediated plant transformation. Front Mol Biosci 3:70. doi:10.2741/4377.

[3] Li X, Thomason LC, Sawitzke JA, Costantino N, Court DL. 2013. Positive and negative selection using the tetA-sacB cassette: recombineering and P1 transduction in Escherichia coli. Nucleic Acids Res. 4122:e204.

We are people of culture too!

Address

Indian Institute of Science Education and Research,
Dr. Homi Bhabha Road, Pashan,
Pune, Maharashtra, India 411008

Contact

P.I Mail: cathale[at]iiserpune.ac.in

Team Mail: igem[at]sac.iiserpune.ac.in

Phone :(+91) 20 2590-8000

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