Question 1

Would any of your project ideas raise safety issues in terms of:

• researcher safety,
• public safety, or
• environmental safety?

Our research is conducted in a safely regulated laboratory, and the ideas of our project do not require etiologic agents, hosts or vectors for experiment. Also no toxic reagent or high risk chemical compound is used. In all, our project hasn’t yet raised researcher safety issues thus far. For we do not apply any virulent genes or etiologic agents, there seems no additional threats on public safety. Yet, the antibiotics resistance plasmid used in our project may harbor the threats of horizontal gene transfer, which will enhance the virulence of other Escherichia coli in the environment. Though a self-destruction mechanism is designed in our project, we are still aware of the risk on environmental safety. The rules and regulations of concern will be under close supervision of Environmental Protection and Occupational Safety and Hygiene Unit of NTUCM.

Question 2

Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes,

• Did you document these issues in the Registry?
• How did you manage to handle the safety issue?
• How could other teams learn from your experience?

The most common question people would have judged us is how we make sure that it is safe to deliver this bug into human intestine. As we noted, the output we would like to deliver, GLP-1, is innate and has no significant harms to the human body. Cell penetrating peptides, which is another peptide we are going to deliver into gastroenterological tract, do no harm as well, since it has a pretty short half life (<15 mins) inside GI tract. What’s more, intestine is an organ with an abundance of normal flora, mainly consisting of Streptococcus and Lactobacillus in the middle intestine. In ileum and section close to ileo-cecal valve, bacteroides and coliform bacteria are dominant. First, for the E. coli itself, it’s very hard to colonize in the GI tract as it would face a bunch of competitors over nutrients and space. Second, the bugs we are going to deliver inside human body is pretty fragile, which can be easily eliminated by some antibiotics, 2nd generation cephalosporin, high ampicillin plus sulbactam, or erythromycin, which is ideal of usage in our cases, since it not only provided anti-microbial effects, but also increases the motility of intestine. Above all, the design we are going to bring to synthetic biology community has little safety concerns, and can be easily circumvented or adjusted.

Question 3

Is there a local biosafety group, committee, or review board at your institution?

• If yes, what does your local biosafety group think about your project?
• If no, which specific biosafety rules or guidelines do you have to consider in your country?

In National Taiwan University, College of Medicine, where the experiments of our project are conducted, Environmental Protection and Occupational Safety and Hygiene Unit of NTUCM takes charge of promoting and supervising safety and health issues. Before beginning of our project, our team members and advisors have attended the safety education series and training courses held by this unit. All of us were qualified by passing the tests at the end of the training to be sure that all the rules of concern are well understood. The application of our project was also verified and qualified. All experiments are conducted under the biosafety rules of Environmental Protection and Occupational Safety and Hygiene Unit of NTUCM, of which all of our eperimental procedures, equipemnts, facilities were under close supervision. http://www.mc.ntu.edu.tw/department/safety/index.htm

Question 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?

As our opinion, we think that for every iGEM teams' circuit design, a mechanism to turn off is necessary. In order to improve the iGEM competition safety concern, we would like to suggest a turn-off mechanism as requirement. Also, the microorganisms should be kept dependent on controlled conditions in the lab to prohibit the unwanted spreading and multiplication outside the lab. Moreover, a suicide switch for all BioBricks or dependence on special nutrients lack in nature for organisms should be engineered in case organisms escape and cause safety concern. The most important thing is that every team and the advisor should know their potential danger and the ways to assess the risk. This aim could be approached by emphasizing the safety rules. Creating a page on the registry for the teams to explain their event tree analysis and fault tree analysis would be an easiest method to increase safety when working with BioBrick parts.

Many turn off strategies have been developed, most of these are the inducible suicide system that can be activated at certain condition. For instance, in our project, we plan to use temperature and small molecule as activating signals( following picture). When the course of treatment ends administration of small amount of tetracycline agonist will induce bacterial death, leaving human body also cause suicide gene activation thereby avoid recombinant strain/gene pollutiing. And splitting suicide system to provide repression in trans can prevent plasmid transfering to wild type strains.

However, these design cannot totally eliminate the risk of horizontal gene transfer(HGT), which recombinant genes can move to other organisms independent of suicide system. So besides suicide system, we come up with a new idea to deal with these kinds of HGT risks by RNA interaction!!!

Expression well designed antisense RNAs have been shown to have inhibitory effect on target RNAs, putting appropriate antisense RNAs on untranslated region of transcripts may interfere target RNA function or translation. This property can be used to prevent HGT. For instance, HGT is more likely to occur between related species, laboratory E.coli have inactivated all its hok/sok toxin-antitoxin system by mutation, but wild bacteria especially pathogenic bacteria have many active TA locus on its chromosome like E.coli O157 have many active hok/sok homologs. we plan to put a stem loop from hok mRNA which can pair with sok RNA 5’sequence on UTR of antibiotic resistance genes we used. IF wild E.coli steal our antibiotic resistance genes and express it, its antitoxin will be competitive inhibited and its toxin will express and kill the thief thus preventing HGT between lab & wild coli. This idea can have wide extension. Besides targeting antitoxin (functional RNA) of type I TA, designing antisense sequences that target RBS to down regulate targeted protein is also possible. Targeting antitoxin of type II TA, essential genes for metabolism, housekeeping genes and any sequences exist in potential HGT receiver but not our coli can be used. Even if the design cannot kill thieves, it can weaken receivers and reduce advantages antibiotic genes bring about thus reduce possibility and danger of HGT. In the past the repression efficiencies of antisense RNA in bacteria are low, but by using the paired termini antisense RNA method and incorporate U turn/YUNR motif, these applications will become more and more feasible.