In our history, our hero is B.subtilis because of capability of catching E.coli, especially gram-negative bacteria species presenting LPS layer with the help of synthesized Limulus anti-LPS factor (LALF) onto the biofilm. On the other hand, in The Canvas Town, all species of our project story including B.subtilis police squad and E.coli gang can synthesize Reflectin protein transferred from cephalopod.
Here, we will answer the safety questions to tell how we will take precautions in our lab experiments and in usage of our project in public.
1. Would any of your project ideas raise safety issues in terms of:
We have one plan to raise safety issues when studying with B.subtilis. Under stress conditions, B. subtilis will form spores. These spores are highly resistant versions of single cells. While B. subtilis itself isn't pathogenic, its spores can be damaging if inhaled. They are also highly resistant to many conditions that are used to kill cells. Therefore, in other iGEM projects, teams in future may want to kill spores to increase their projects’ safety level. To achieve this, we optimize a procedure to kill spores in liquid cultures and on solid surfaces with the help of aqueous dissolved Oxygen, Ascorbic Acid, and Copper Ions involving treatment, whose details are given in human practice part.
Researcher Safety
Our all experiments have been performed in the Synthetic Biology lab, opened for following iGEM competitions at Fatih Medical School. During project, we have made lab meetings in a week to consider safety rules and lab regulations before starting to our project. Also, during lab experiments, lab technicians and instructors of our team have been helping in handling and safety removing of B.subtilis and E.coli bacteria culture. Also, project advisor has been participating in all lab executions to perform risk assessments which may rise during experiments and lab usage. In addition, we plan to design a biofilm containing anti-LPS factor even without B.subtilis and its spores in our project. Because of that reason, we will also kill B.subtilis spores on the biofilm before testing our biofilm with exposure to E.coli by performing dissolved oxygen procedure. This procedure is optimized to kill the spores which may be infectious to humans when using our designed biofilm on the surfaces. Thus, we try to maximize safety of our project. Next, we add some general safety issues related with our studies in our lab as follows:
Some safety Issues:
1. What protective equipment will be used to minimize exposure of laboratory personnel who will be working with the agent? lab coats and gloves
2. What will be used for handwashing? Soap or alcohol gel
3. Please state the signs and symptoms of infection. Ingestion of E. coli may cause diarrhea with or without abdominal cramps. Skin infections may be inflammed. Conjunctivitis may occur after inoculation of the eye.
4. Please describe post-exposure therapy to be used in the event of accidental exposure. Rinse contaminated areas with soap and water. Mucous membranes should be flushed with water. For accidental ingestion rinse mouth with water, but do not swallow
Actually, in lab procedures, there were no many chemical hazards. In gel preparation and electrophoresis, there were some dangerous chemical but these procedures were being performed in the special electrophoresis room and its responsible team members were potentially informed about the hazards.
On the other hand, in other procedures, we have had some biological hazards especially related with B.subtilis. Throughout the project, we used the Escherichia coli strain Top10 and BL1; Bacillus subtilis strain 3610 and Bacillus subtilis strain 168. Wild-type E. coli is classified as a hazard group 2 pathogen by the UK Advisory Committee on the Dangerous Pathogens (ACDP). Wild-type Bacillus subtilis (i.e. strain 3610) is classified as an ACDP hazard group 1 organism and its derivative B. subtilis strain 168 has disabling auxotrophs mutations (e.g. conferring a requirement for tryptophan, Zeigler et al, 2008) that makes it even less likely to colonise or cause harm to human or animal health. B.subtilis is at level 1 biosafety according to World Health Organization (WHO).
Public Safety
To increase public safety of our project, our first plan is to kill all infectious particles especially B.subtilis spores on the biofilm. To do this, as we mentioned above, we clean the biofilm from the spores by the treatment with Fenton reagent containing dissolved oxygen and other constituents. Thus, our designed biofilm will be safe for public use and its effectiveness and safety level can be improved in future.
B.subtilis is not associated with disease process in humans, however, may prove to be opportunistic pathogens capable of causing infection in the very young, in the aged and in immunodeficient or immunosuppressed individuals when we regarding public safety. Before using our project in public condition, especially on food safety and non-pathogenic surface safety via using biofilm, considerable further research and tests are absolutely necessary as our Fenton reagent treatment protection. Indeed, production of biofilm from B.subtilis without B.subtilis bacteria and only including anti-LPS factor is necessary and should be such a non-pathogenic biofilm. To be able to remove pathogenic effects of B.subtilis and its waste is necassary before using on surface to be bacteriacidal for gam-negative bacteria via anti-LPS factor for public safety.
Environmental Safety
Bacillus subtilis strain 3610 is wild type strain that is already widespread in the environment. It has not been modified to enhance their ability to survive, disseminate or displace other organisms. Thus, there is no specific environmental hazards associated with the Bacillus subtilis strain 3610 except its spore formation. Environmental safety is kept to prevent from spores in our synthetic biofilm by Fenton reagent treatment.
E. coli strain TOP10 and BL1 have very limited ability to survive outside the laboratory so that it would be unable to survive, disseminate, or displace other organisms in environment. Therefore, there is no specific environmental hazards associated with the E. coli strains.
Bakterýlerýn atýlmaýs
2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?
A.Did you document these issues in the Registry?
We have documented our safety issues possessing device with the part ID Bba_K541001. The device which produces and secretes Limulus anti-LPS factor (LALF) into medium by B. Subtilis, which can bind LPS on gram negative bacteria and neutralize its biological effects or enhance its clearance could have important clinical applications. Lipopolysaccharide (LPS), or endotoxin, is the major mediator of septic shock, a serious complication of Gram-negative bacterial infections in humans. Thus, this part can be used to kill infectious gram negative bacteria in iGEM projects if needed like antibiotic.
Also we will add alternative devices and parts to raise safety issues in the registry.
B.How did you manage to handle the safety issue?
In our Project, we plan to design LALF protein producing system in a specific bacteria that will not be harmed by this bactericide itself. To do this, we needed a bacteria type that has no lip-A structure which is bound by LALF. Thus, we decided to use Bacillus subtilis colonies which are the most prefered gram positive bacteria in iGEM. In conclusion, our “protector” bacteria affects on E. Coli mortally, therefore our remaining region is cleared from E.coli. As a future aspect, a special covering material, a biofilm for example, may be used as a cover sheet on surfaces that leads to protection of bacteria.
C.How could other teams learn from your experience?
Other teams can have knowledge about our experiences with anti-LPS factor synthesized from B.subtilis against E.coli via our wiki, especially in project plan site.
Into iGEM forum system, we have opened a box for `The safety parts` under `lab’ main box (https://igem.org/forum/Comment_List?n=322&s=1); thus, teams can learn from the experiences of parts of other teams. Also, we can add safety issues video via Youtube and integrate into the forum to inform other teams.
3. Is there a local biosafety group, committee, or review board at your institution?
4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?
We think that Synthetic Biology Safety Police Squads can be founded for each continental competitions. This squad may be chosen from an iGEM team from each continent or colloboration of some experienced teams or independent individuals. Firstly, an iGEM team from a continent wanting to be the safety squad, can control new established synthetic biology labs and perform meetings via electronic communication ways (i.e. Skype) to share their safety precautions with new iGEM teams about their lab needs for security and improvement of project safety of new iGEM teams. For example, experienced iGEM team which may be also experienced with viral Synthetic biology projects can help another iGEM teams which possess to perform synthetic biology projects on viruses. Thus, experinced teams can collaborate with new teams their experiences while doing such projects. Thus, the Synthetic Biology Safety Police iGEM team can be also regarded as completing their ‘Collaboration option with other teams’ which is required for Gold Award in judgement.
In addition, a safety plan, procedures, prerequisites and meeting how to perform a safe iGEM project in a lab can be gathered in a CD which may be distributed to each iGEM team with Part Distribution. Thus, each team can update their labs and their project thinking ways during studying time. |
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