Following iGEM's rules and policies
1.Are you planning to do any of the following activities,which are prohibited in the competition?Check all that apply.
No,we are not planning to do any prohibited activities.
2.Please read over iGEM’s White List.Will your team use any organisms or parts not on the whitelist,or do any activities not on the whitelist?Check all that apply.
No,all our work is covered by the White List
3.Are you planning to do any of the following activities that require advance permission from iGEM?Check all that apply.
No,we are not doing any of the kinds of work outlined above
4.Are you collecting any data about people,such as their opinions,quotations,medical history,gender,behavior,attitudes,or concerns?
Yes,and we have confirmed that relevant laws,regulations,and institutional rules do not require us to get formal approval
About your lab
5.Please upload a photo or two of your lab showing the relevant safety features.
No,we are not planning to do any prohibited activities.
6.What is the biosafety level of your work space?
Level 1 – standard microbiological lab
7.Which work areas will you use/are you using to handle biological materials?Check all that apply.
(1) Open bench
(2) Chemical fume hood
(3) Other(ultra-clean-workbench)
About your project
8.Describe the goal of your project:what is your engineered organism(or other synthetic biology product,system,or tool)supposed to do?
We want to use synthetic biology to enable strictly anaerobic bacteria Clostridium tyrobutyricum to gain oxygen tolerance,and be capable to grow well under aerobic conditions.
Endogenous type I-B CRISPR-Cas system is used to knock out the perR gene from the genome of C. tyrobutyricum strain L319 and construct it in plasmid pMTL82151,using engineered anaerobic-inducible promoters to regulate its expression.
9.Which whole organisms,including viruses and cell lines,will you engineer in your project?Check all that apply.
Escherichia coli(DH5-alpha,BL21,CA434,JM109,MG1655)
Others (Clostridium tyrobutyricum L319)
10.Will you use any other organisms in your project?
Clostridium acetobutylicum ATCC 824
11.As part of your project,are you planning to make/have made new parts or substantively changed existing parts in the Registry?
Yes
12.Could any of your parts be hazardous on their own and/or in the context of your project?
None of our parts could be hazardous
13.What experiments will you do with your organisms and parts?
Almost all plasmids we constructed in out project are based on pMTL82151,which is a kind of widely used Clostridium spp.-Escherichia coli shuttle plasmid.
The process of introducing engineered plasmids into C. tyrobutyricum requires a conjugation process with Escherichia coli CA434.
At first, the perR sequence on the genome of C. tyrobutyricum L319 is knocked out by introducing an engineered plasmid constructed with an Endogenous Type I-B CRISPR-Cas System into the wild strain. We plan to construct it into a plasmid, using engineered promoters to regulate its expression.
Then, we synthesized the sequence of the vgb promoter and characterized it using fluorescent proteins BS2.
Finally, we will rationally design the promoters to obtain engineered promoters with better anaerobic induction effect.
14.What kinds of chemicals are you using in your project?
(1) Carcinogens
(2) Highly flammable chemicals
(3) Acids and corrosive chemicals
Most of the hazardous chemicals are reagents used in SDS-PAGE,as follows:AM(Acrylamide),APS(Ammonium persulphate),and TEMED(N,N,N',N'-tetramethyl ethylenediamine).
Identifying project risks
15.What hazards are presented by the organisms,parts,chemicals,or experiments you described in Part 2?Check all that apply.
Environmental hazards
Our transgenic bacteria may lead to resistance gene transfer,affecting the growth of wild-type bacteria.The media carrying antibiotics in the experiment are harmful to the environment and may alter the composition of environmental microorganisms or promote the emergence of super-resistant bacteria.
16.For each of the hazards you identified in the previous question,please give 1-3 sentences describing how harm could occur.
The engineered bacteria might escape from the laboratory accidentally,like flowing into the sewer without sterilization,or being carried out by someone in the lab that accidentally did not sterilize their hands or clothing before leaving the lab.
Although Clostridium tyrobutyricum strain L319 and Escherichia coli K12 strains are considered safe for human beings,but if the engineered C. tyrobutyricum and E. coli escape from the laboratory into the environment,they may affect the survival of wild bacteria in normal environments(such as sewer or animal gut)and aerobic environments.
Anticipating future risks
17.Imagine that,in the future,your project was fully developed into a real product that real people could use.How would people use it? Please describe the other future use and how your project would be used in the real world?
If our project could be fully developed,we hope this technique could be used on alien planets for the use of soli improvement.
When humankind land on distant planets(such as Mars),they need to establish bases and improve the soil to adapt to the planting conditions,and using microorganisms for soil modification is naturally a good choice.
The landing of humans and the cultivation of plants will bring an aerobic environment,so the bacteria used in soil improvement may also need to adapt to that condition.
When we make the strictly anaerobic Clostridium tyrobutyricum gain the ability to grow under aerobic conditions,this technique or method could be used on other bacteria,then they could be adapted to extraterrestrial planet soil improvements without losing its effect in the presence of oxygen.
This could provide support for the future explorations on other planets.
18.If you were permitted,would the continued development of your project require release beyond containment?
Yes,open release in the environment.
After the project is completed,if we do test the effects of soil improvement,we will consider small-scale experiments on a small area of land which conditions are close to the surface of the alien planets.Though the oxygen tolerance tests may be performed in the laboratory rather than in the environment.
If our project progresses to that extent,we must follow Biosecurity
Law of the People's Republic of China.
(http://www.npc.gov.cn/npc/c30834/202010/bb3bee5122854893a69acf4005a66059.shtml)
19.Have people on your team had a conversation(within your team or with someone outside the team)about how any of the bad outcomes below might relate to your project?Check all that apply.
Harm to human health and safety
20.Considering the future use(s) and conversations from the previous questions,do you think your project could potentially lead to any of the bad outcomes listed below?Check all the appropriate boxes and expand in the comments section.
Harm to human health and safety
Harm to the environment,including wild plants and animals
21.How might the bad outcomes that you identified in the previous question come to pass?Check all that apply.
Accidental exposure to a hazardous organism or chemical
Accidental release of an engineered organism or part into the environment
22.If your project were fully developed,could any of your engineered organisms or parts spread autonomously in the environment?
Yes,autonomous environmental spread of one or more of our organisms or parts is possible.
Due to the difficulty of constructing kill switches in Clostridium tyrobutyricum,and considering the negative effects of protein toxins,we chose to prevent the spread of our engineered bacteria through strict sterilization treatment.
Even so,the engineered Clostridium tyrobutyricum might escape from the laboratory accidentally,like flowing into the sewer through water without sterilization when there's a severe mistake in the operation.
Managing risks
23.Who are the experts,other than your supervisor(s),supporting you in managing risks?If you discover a hazard or risk in your project,who would you go to for help?
In our team,Dr. Zhengming Zhu is the Principal Investigator whom we consult for laboratory safety and procedures.
We can also seek help and guidance from our institutional biosafety officers,they are all familiar with laboratory safety procedures in genetic engineering and will ensure the proper use and disposal of all laboratory materials.
24.What safety and security rules or guidance cover your work?
Biosecurity Law of the People's Republic of China is the most important reference law.
(http://www.npc.gov.cn/npc/c30834/202010/bb3bee5122854893a69acf4005a66059.shtml)
At Nanjing Tech University, the Laboratory Construction and Management Department(http://sys.njtech.edu.cn/)is responsible for the construction and management of the entire laboratory,laboratory safety,and environmental protection,experimental equipment purchase,and user management.
Measures for the Management of Experimental Safety Risk Assessment of Scientific Research Projects of Nanjing University of Technology(http://sys.njtech.edu.cn/info/1087/3361.htm),together with Technology Laboratory Safety and Environmental Protection Management Measures of Nanjing University(http://sys.njtech.edu.cn/info/1087/3105.htm)are the institutional safety and security rules that we followed while carrying out our lab works.
25.Will your project need extra support or review to manage the risks you have identified above?
Yes,if our project were to be developed further for a real-world use after the iGEM project stage
26.Have your team members received any safety and/or security training?
Yes,we have already received safety and/or security training.
27.Please select the topics that you learned about(or will learn about)in your safety and security training.
Lab access and rules
Responsible individuals(e.g. lab or departmental specialist or institutional biosafety officer)
Differences between biosafety levels
Biosafety equipment(e.g. biosafety cabinets)
Good microbial technique
Disinfection and sterilization
Emergency procedures
Rules for transporting samples between labs or shipping between institutions
Physical biosecurity
Personnel biosecurity
Data biosecurity/cyberbiosecurity
Dual-use research and/or experiments of concern
Chemical,fire and electrical safety
28.What laboratory biosafety and biosecurity measures are you using to manage the risks in your project?
Accident reporting
Personal Protective Equipment/PPE
Inventory controls
Physical access controls
Data access controls
Lone Worker or Out of Hours policy
Medical surveillance
Waste management system
29.What other actions have you taken to manage the risks in your project?
Our laboratory have strict rules about biological safety, like biosafety management and garbage classification. We all have passed the safety training exam held by Laboratory Construction and Management Division of Nanjing Tech University and we believe that our team and our project is qualified to perform these experiments safely.We have learnt the key information about biosafety and biosecurity from other team member who participated in the Values and Risks workshop held by iGEM community.We have learnt about the countermeasures of out engineered bacteria, parts, and future products from Dr. Zhengming Zhu, and learnt the biosecurity handbook of Nanjing Tech University. We are trying to reduce the total risks of our project,so we use attenuated strains like E. coli K-12,and we well carry out all biocontainment measures we could to achieve that.
30.Overall,how will the actions you’ve taken,expert support,rules,training,and other procedures and practices you described help you to manage the risks in your project?
Those who fail to pass the laboratory safety examination shall not enter the laboratory to carry out experiments. One must comply with the provisions of the laboratory and strictly implement the safety operation procedures. Students must be familiar with various hazard sources in the laboratory,and master protective measures and emergency disposal methods. Wear appropriate personal protective equipment when entering the laboratory. There are no objects or behaviors irrelevant to the experiment in the laboratory. We have access to a biosafety level 1 laboratory and are able to conduct our experiments in open benches and ultra-clean workbenches. The laboratory have established a safety duty system.The personnel who leave the laboratory at the end of each day shall check the water and electricity safety,then make records. Sort and dispose garbage properly. Waste classification includes gloves and soft wastes,waste glass,waste liquid,solid wastes and sharp objects.After the experiment,the waste will be classified and disposed.