Contents 1 Overview 2 2012 Calgary 3 2012 Paris Bettencourt 4 2012 Colombia 4.1 Ideas of the project 4.2 Safety concerns of their project 4.2.1 (1)Biosafety Provisions 4.2.2 (2)Safety Considerations: Hazardous reagents and Biohazards 4.2.3 (3)Biological Parts and Devices 5 2011 Imperial College London 6 2011 IIT Madras 7 2011 Gaston Day School 8 2010 SDU-Denmark 8.1 Ideas of the project 8.2 Safety concerns of their project 8.2.1 (1)Project and researcher safety 8.2.2 (2)Laws and guidelines 8.2.3 (3)Watermarks 9 2010 VT-ENSIMAG Biosecurity 9.1 Ideas of the project 9.2 Safety

Overview

In our project, not only do we like to develop a peptide delivery system “PEPDEX”, we also try to apply the system to solving medical problems since we are medical students. Though we have discussed a lot about the safety issues in our project, designed different ways of regulation (even suicide) to avoid spreading out of the lab or contaminating the environment, we would like to know what do medical doctors think about biosafety problems in synthetic biology. Therefore, before the interview with doctors, we had collected the safety information from several teams that won the safety commendations from 2010 to 2012. During the interview, we demonstrated how iGEMers deal with biosafety problems and get feedbacks from the doctors.

So, how do these teams assess the risk of these new BioBricks or genetically modified microorganisms? Lab safety courses were common among iGEM teams before entering laboratories. Consulting biosafety group representatives was adapted by some teams, too. There were several teams that followed the guidelines or laws of GMOs (genetically modified organisms) in their own country, and modified them into a new guideline for future iGEM teams. To monitor or to avoid spreading of BioBricks into environment, the methods showed even more diversity: adding a barcode or watermark that can be easily identified by others, using toxin-antitoxin system to avoid the transformation of plasmids into other organisms, or creating a new way to select plasmids without using antibiotics…..etc.

The details of “safety in iGEM” are shown below. And to see feedbacks of the doctors about biosafety of our project, see Doctor Interview

2012 Calgary

In their project, they use two genetically modified products, FRED and OSCAR, to detoxify tailing water. They illustrate that there are four common major safety concerns with their project:

1. Engineered bacteria may be harmful to the environment
2. The engineered bacteria may outcompete the naturally occurring bacteria
3. Gene may be transferred from synthetic bacteria to natural organism
4. The genetically engineered bacteria may undergo mutation and evolution.

They design the mechanical engineered controls and biological engineered controls to solve the problem.

For the mechanical engineered controls, the final product containing FRED will be made within one-time use closed containers, with one-way valves. The bleach will be released to the operator to destroy FRED prior to proper disposal. Also, OSCAR is designed to work in a closed system. But it may escape by adhering to the belt. To counter this issue, they treat the belt with UV irradiation as it exit the bioreaction solution, which will destroy OSCAR without affecting the generated hydrocarbon.

For the biological engineered control, a “Ribo-Kill-Switches” system is introduced to the design, which contains micrococcal nuclease and CviAII restriction enzyme and will initiate cell death through degradation of genomic and plasmid DNA. Once the bacteria escape the closed bioreactor, the biosensor will turn on the suicide gene and destroy the engineered bacteria.

Besides the above effort mentioned, their participants are also well trained and their experimental procedure are safely conducted as well. The iGEM 2012 Calgary team is required to attend biosafety courses and Workplace Hazardous Materials Information System (WHMIS) training sessions. Their bacterial strains and samples all follow biosafety regulation and Material Safety Data Sheets (MSDS). They all use safe, non-pathogenic bacterial strain of E. coli for gene-characterizing and gene-testing, which is commonly used in lab worldwide.

2012 Paris Bettencourt

The main goal of Paris Bettencourt iGEM 2012 team is trying to overcome the safety concern on horizontal gene transfer. Many iGEM team had promote ingenious ideas among various fields of synthetic biology application, however many projects also face the problem of horizontal gene transfer if applying to the real world.

Therefore, Paris Bettencourt 2012 team come up with a project, bWARE, to tackle with the risk of horizontal gene transfer. Their design consists of three major mechanism in order to deal with this obstacle; and they chose AgrEcoli, a nitrate biosensor, as their hypothetical case study material.

The first part of their safety design is a physical container called alginate capsules, a type of polymer gel. It aims to totally entrap the bacteria within it, so there will be no iGEM designed bacteria released to the environment. The second design is a semantic containment, which prevents gene expression in wild type bacteria. Paris Bettencourt iGEM 2012 team used an amber codon embedded in protein genes to prevent their expression in wild type bacteria and an amber suppressor system in their genetically engineered bacteria to maintain its targeted function. The third part of it is a delayed population-level suicide system through a complete genome degradation of the whole population. They coupled their toxin anti-toxin suicide system with a delay system, which will allow the cell to perform its intended function before the DNA-degrading suicide machinery is expressed. By realizing the gradually dilution of regulatory transcription factor and the special use of stationary-phase specific promoter, they can delay the activation of the suicide system composed of a restriction enzyme and an anti-toxin plasmid, which together will destroy the whole engineered genome when the time comes.

With these three levels of safety design, their studies and wet lab experiments showed convincing result that many of their design do work; and may play an important role in improving the applicability of iGEM genetic engineered bacteria by reducing the risk of horizontal gene transfer.

2012 Colombia

Ideas of the project

The extensive use of fungicides and pesticides to control field epidemics in Colombia has raised an issue of the poisoning effect to public health. To solve the problem, this team modified a bacteria that can detect pathogen associated molecules, amplifying the signal, and eventually stimulate the hypersensitive response (secretion of salicyclic acid in this project) of the plant to defense infection.

Safety concerns of their project

(1)Biosafety Provisions

General regulations of the University and safety guidelines for laboratory practices were followed. methods and safety precautions are well within the regulations of the Committee. All team members were required to attend safety training.

(2)Safety Considerations: Hazardous reagents and Biohazards

For health risk and personal safety, safety courses were required before the team members enter the lab. Bacteria were sealed completely during culture, and plants were inoculated at isolated greenhouse. Bacterial strains used in their project, Escherichia coli K12 does no harm to human and cannot survive in the environment other than laboratories in the long run. Aliivibrio fischerii is not considered dangerous to humans, either. Ralstonia solanacearum, though pathogenic to plants, were well handled by qualified and experienced personal in flow chamber. They had also included a way for the bacteria to control their own population density using a Toxin-Antitoxin module (TA). As with known toxic chemical reagents and hazardous physical agents were managed according to the guideline, and the members were well-informed about the toxicity of these agents.

For public health, the adverse effect of salicyclic acid and its metabolites were listed, but it may do no harm to human because it would have bactericidal effects before reaching concentration that causes toxicity. Moreover, it is well-controlled by negative feedback.

For environmental qualities, These sequences have been previously characterized. To avoid cloning unknown pathogenicity, they amplified the majority of genes from the start codon to the stop codon, and a Blast was carried out to confirm the results.

Last but not least, they made sure that the circuit is tightly regulated by positive and negative feedbacks, and the production of salicyclic acid is only induced by pathogen associated molecules but no constitutively expressed.

(3)Biological Parts and Devices

Some of the genes they used are involved in the sensing system, but they also may be related to pathogenicity processes. However, neither of them can cause disease by themselves, as they are not toxins. All the information about these genes is read by the team members, too.

2011 Imperial College London

The team Imperial College London wanted to protecting the soil from erosion. To achieve this, they design bacteria that would secret IAA, a hormone which would promote root growth. As the root growth faster after the seeds germinate, the ability for plants to hold soil will be enhanced, thus helping anchor the soil.

Since they’d like to introduce genetically modified(GM) bacteria into environment, they take the safety issue seriously. First, in their system, they design “Gene Guard” to prevent horizontal gene transfer. They engineered anti-holin into the genome, and holin and endolysin on plasmid. If horizontal transfer happens, accepted bacteria don’t have anti-holin to neutralize holing thus will be killed. Second, to make sure their project is practicable and safe, in human practice they have deal it from many prospect, including searching laws about GMOs. They also consulted many professors, such as social scientists, environmental scientists, plant experts and so on to ensure the safety of their idea. Last, they made a GM release guideline for future iGEM teams who may cope with the same problem. They aggregated their experience and wrote down many questions we need to carefully think about, and give some optional solution for them. This gives convenience to teams facing this situation.

2011 IIT Madras

Team IIT Madras made a revolution on the selection method we use today. Instead of the traditional idea that use antibiotic resistance gene, they replace it by Proteorhodopsin(PR), a green-light driven proton pump. Cultured in minimal media, PR expression will provide energy advantage to plasmid owner, for PR gives the ability to use light as an energy source. Thus make a selective advange.

In this way, they overcame some disadvantage of using antibiotics. For example, avoiding the risk of horizontal antibiotic gene transfer and spread. Besides, this could reduce use of antibiotics on synthetic biology, because antibiotics are in fact toxins to the environment. And this would make a more environmental-friendly future.

Since Proteorhodopsin uses retinal as a chromophore for light reception, the supplied retinal is a key protein for the growth of the bacteria, hence people could give control to this system.

2011 Gaston Day School

Team Gaston Day School try to build a nitrogen detector that is easy to use for needed people, like farmers and ranchers. They use RFP as reporter, thus simple to know if there is nitrogen pollution.

After the detection, the bacteria have to be destroyed in case the drug-resistant ability spread out. Rather than using complicated circuit or method, they choose to carry out a simple method – bleach. Amazingly, bleaching has a significant effect on killing bacteria. It seems can kill the bacteria completely. Once we follow the bleach direction could we use the bacterial detector or other modified bacteria safely.

They give us an example that we don’t always have to find out the solution from a sophisticated way. Sometimes simple, is the best.

2010 SDU-Denmark

Ideas of the project

The project of SDU-Denmark is “Flow-E”, which is a nano-scale machine that generates a flow by a bacterial pump. The bacteria were aligned along the internal surface of a mirco-tube, and the flow was generated through the synchronized movement of the flagella of the bacteria. The flow depends on the number of the flagella, and to alternate the flow, synthesis of the flagella was regulated by the intensity of light.

Thus, the project may be divided into three parts: retinal biosynthesis, phototaxis by photosensors and hyperflagellation, which will be combined into bacteria eventually. Phototsensors are activated by the light, and the signal transduction pathway was then started and linked to the synthesis of flagella, resulting in hyperflagellation. Retinol acts as a relayed component that initializes the signal transduction.

Safety concerns of their project

The safety concerns of the project was divided into three parts: (1) project and researcher safety (2)laws and guidelines (3)watermark.

(1)Project and researcher safety

During the election of the project, numerous ideas were considered by the team: the environment, foods, health & disease, physics/chemistry/biochemistry. First of all, they discussed about which bacteria it was possible to use. Medical ideas for implementing in the body were also considered. Secondly, which type of the actual mechanical work to be done tended to be a great issue. Pili were considered but then abandoned due to its virulent characteristics in some bacteria, which may lead to pathogenic consequences (ex: using pili of Pseudomonas aeruginosa, which is pathogenic to human body)

E.coli were considered because they are very well adapted to the laboratory environment, are easy to keep alive, can be fairly easy modified, and they no longer have the ability to thrive inside the intestines.

Furthermore, they have used their risk-assessment guidelines to describe the safety of each of our contributed parts, which included four dimensions and several related questions:

General use-What type of lab should work with this BioBrick be done in?......etc.

Potential pathogenicity-Does this BioBrick produce anything that plays an significant role in environmental processes?…. etc.

Environmental impact-Does this BioBrick produce anything that plays an significant role in environmental processes?...... etc.

Possible malign use-Can your chosen host be arosoled? Does this BioBrick increase the host’s ability to vaporize?……etc.

Each part was accomplished by BLASTed the sequences of their parts, searching for the function and homologs of the pars, considering and assessing the questions mentioned earlier. Among all parts, only hyperflagellation part was considered to increase the pathogenicity of bacteria. However, the bacteria they chose are unable to survive and reproduce outside of laboratory conditions.

(2)Laws and guidelines

Risk assessment

Based on the current UN and Denmark rules and law for genetically modified microorganisms (GMM's), risk-assessment was developed by listing some questions as assessment factors for evaluation, so that they can judge if our use of GMM's poses a threat towards the well-being or safety of human beings, animals, plants, or the environment.

Substitution

In a suitable system, compatible with the intended work, that is safer for humans, animals and plants, or the environment is always significant, which might be achieved by substituting the other, more dangerous system. In their study, though the E.coli strain they used (MG1655 and TOP10 strains) was harmless, issues of substitution was also taken into consideration due to the laws and guideline in UN and Denmark.

Assessment by local bio-safety group

Al the members of their team attended laboratory safety course from the Committee for Students’ Laboratory Safety to ensure everyone knew basic safety precautions before entering laboratories. For their project, all the experiments were done in security level 1 laboratories, and no pathogenic bacteria were used.

They also had interview with a representative of “Arbejdsmiljogruppen” , the local bio-safety group associated with the University of Southern Denmark. During the interview, they explained the details of the project, asked several questions about the biosafety(ex: if there would be increased risk due to work performed by relatively inexperienced students, or if they perceived any danger should the bacteria get out of the lab……), and get the replies about the safety from the representative to ensure the biosafety of their project.

(3)Watermarks

Watermarks are special sequences that were like a license. They are easy to find, easy to read and easy to insert by the developing team. They must be persistent in the plasmid, in order to recognized by other researchers in case it contaminates the environment or are accidentally transmitted to other labs. In conclusion, they chose to divide the code into two separate parts, each is from 6 to 26 nucleotides. Two equally long parts at each end of the coding sequence ensures symmetry , which again should help make insertion of a watermark easy, as this will reduce the complexity of the primers we need to design to insert them.Moreover, this ensures that identification is done easily, since the combination of nucleotides of the sequence from E to X and from S to P is known.

2010 VT-ENSIMAG Biosecurity

Ideas of the project

This project “GenoTHREAT software” is a sequence screening tool that can be used to detect potential select agent and toxin. Based on the “Screening Framework Guidance for Synthetic Double-Stranded DNA Providers" in late 2009 by the U.S. government, which focuses on minimal DNA sequence screening protocol, this team developed their own general algorithm for each input sequence.

For the general algorithm, initial nucleotide strand and its reversed DNA sequence are kept, and six frame translation is done. These subsequences are then blasted. According to the blasting result, suspected toxic sequences are picked, where relevant information is acquired. The information includes some keywords, like the strain of the pathogenic bacteria that produce the toxin, the names of diseases associated with the entries……etc. It is then checked by their database derived from CDC Select Agent and Toxin List to see if it is consider to be dangerous. Finally, if one subsequence leads to a hit, then the initial Query Sequence is a hit.

To further optimize their software, they have put intervening sequences, random mutations, degeneracy to test the sensitivity and specificity. In practice, they screened all the registry of iGEM and gave an analysis of the result.

Safety

Actually, this project focuses on biosafety from head to toe.

First, it is very persuasive since they didn’t construct any BioBricks, which won’t pose any threats to the environment. Secondly, they also used the reconstruction of the 1918 Spanish flu virus in 2005 as an example to disclose the “double-edged sword “characteristics of synthetic biology, thus put more emphasis on the importance of “GenoTHREAT software” as it can rapidly screen any potentially harmful agents.

Last but not least, they have put it into practice by screening all the registry of iGEM. It showed that after screening the first 1724 first sequences of this registry, the hit rate was reduced from 6.5% to 2.9% by altering certain program parameters. Following further analysis they also ended up with one true hit: the sequence BBa_I10020, which was already claimed dangerous by iGEM. These studies thereby give a strong proof of the accuracy of “GenoTHREAT software”.