Safety
Safety and security can't be more important and they should be branded in every team member's mind. Apart from the safe lab practices and operations, bio-safety to avoid leakage, release or misuse of the engineered organisms is also one of the core concerns of iGEM.
This year, in order to ensure the bio-security of our project, we have made careful assessments and adjustments to each step. Related strategies and considerations are listed below, from project designing early this summer to the questions potentially emerging in the future implementations.
See our work from the following aspects！
Safe operations in the lab is educated to evey team members, no matter ultimately taking part in the wet-lab or not, by the lab manager. Commonly ignored questions such as balancing the centrifugue before rotating, closing UV light before using a bench, monitoring the microoven when heating and releasing extra water from the autoclave were carefully emphasized.
Besides, every protocol in our experiments will be carefully checked and disscussed by wet-lab members to assess its feasibility and safety. Our instructors have supervised our procedures before and when we conducting the experiments.
B.subtilis was chosen as our chassis not only for it is a commonly-used, well-characterized model organism easy to be engineered, but also it is a safe and Risk Group 1 organism that is non-pathogenic and no harm to human. However, since B.subtilis is a spore-forming organism, a check-in form has also been submitted by us to report the usage!
Besides, since we were doing project with stabilized spores, biosafety cabinet was used when we purified spores from sporulation medium as well as in other operations with spores.
In our experiments, we increase the resistance of B.subtilis to radiations. In response to the concerns of the stronger resistance which, if released accidentally, may potentially lead to safety issue, we take some measures to manage it:
1. The special substances which arm our spores are transferred in plasmid pHT01, which express exogenous proteins in an IPTG-induced manner. By undergoing some testing experiments, without IPTG, little expression leakage has been discovered. Besides, a promoter with more adaptations and less leakage can also be used in the future experiments.
2. One of the substance we employ to resist radiation is the melanin, which has been used in many previous iGEM teams for similar purposes. The synthesis procedure for melanin needs L-DOPA or tyrosine as substrates, which is not normally existed in a reaction-inducible manner in the natural environment. Therefore, without substrates adding externally, no melanin can be produced and less resistant is the spore in natural environment.
3. We only increase the resistance of spores to radiations but not to any other factors such heating or high pressures. Therefore, our B.subtilis can be normally killed by those commercially available antibiotics, and the method of autoclave can still effectively kill the bacteria and spores alike. Related experiments have been done to testify the efficiency of common disinfection protocols (antibiotics and autoclave).
4. B.subtilis is in Risk Group 1 and is a well-characterized, safe organism to mankind. Besides, amid our project, no hazardous engineering has been done.
5. In our implementation, we plan to put the spores in a container in the cargo ship, where temperatures and pressures can be much harsher than normal conditions in which the spores germinate. Besides, if our spore released in to the vacuum space without any protection, it shall most likely to be killed by many factors even beyond considerations.
6. Last but not least, we plan to carry out experiments with B.subtilis strains whose key genes for gemination to be totally knocked out, thus largely prevent the contaminations. Based on this, a kill-switch which shall induce the synthesis of toxic molecules for B.subtilis after spore germination may be designed in the future.
In order to address the potential anti-microbial issue, we consult two experts in this field, we have already introduced the meeting between us in the integrated human practice and the followings are the main content focused on the safety issue.
Prof Lei Rao is working in the field of how to kill spores. He introduced the major ways that scientists are using today to remove spores. He pointed out that heat and high pressures are still the most effective, practical and popular way, while sterilization by ionizing radiation is limited in some application scenario such as the food industry. Concerning the question that whether our spores can be a potential risk when release to the space, prof Rao did not think so. If it is exposed to the vacuum alone without any protection, he added, it would eventually be eliminated with very few, almost none, keep vitality to make substantial damage.
Dr Shen told us about the topic of Planetary Protection, which is one of the major topics when astronomers talking about Mars or icy moons, and it is also targeted spore-forming organism such as Bacillus. Nowadays, spacecrafts that head for Mars have to be rigorously sterilized to ensure that no more than 10 spores per square meter on the outer surface.
Additionally, he suggested that high concentration of formaldehyde can kill almost all the spores, but the sophisticated spacecraft may not allow this operation, so there will always remain some unkilled microorganisms. Under this circumstance, sterilization method with ultraviolet irradiation is also difficult to completely sterilize some parts. However, maybe we can try to use formaldehyde for sterilization in the lab with doors closed and acetic acid sprayed to eliminate the toxicity.
We had a meetup with Beijing Institute of Technology to discuss bio-safety since we, as well as SJTU-software are all concern about it especially we're working on spore-producing organism.
To learn more about our meetup on the safety issues, please visit collaborations.
Through public survey in the early stage, teams from SJTU-BioX-Shanghai and ShanghaiTech have found that the public is confused but interested in astrobiological safety issue, such as planetary protection and so on. We conducted the survey because both of the teams are working on astrobiology so we've determined to introduce an open course on the bio-security to the public.
To learn more about our meetup on the safety issues, please visit collaborations.