Team:USTC-China/safety

Project

Predecessor of our project

When we were still in the stage of brainstorming, we came up with a project about defeating the AIDS, which is the predecessor of our project. We planned to inhibit the virus from delivering its genome into the host's nucleus by means of using aptamers to bind specific sites on the outer nuclear membrane. However, many questions concerning about the safety of the researchers, the public and the environment made us give up this idea.

The researchers:

First, our team members have no experience in studying the AIDS and don't know the proper methods of managing this virus. What's worse, there are no laboratories in our institute study on the AIDS and we have no resources to get necessary trains.

Second, our lab cannot provide enough protection to our members. The condition of our laboratory is far from reaching the standards for a lab to study the AIDS. For instance, our lab cannot be divided into clean areas, polluted areas and semi-polluted areas. We do not have specific equipments for sterilization aim at AIDS.

The public:

If we chose the project of AIDS and the virus infects the researchers through body liquid, then there would be accesses for the virus to the public.

The environment:

Although the AIDS cannot spread by air, water, drainage system or social contacts, there are still possibilities for the viruses which are released and survive in the environment to mutate and get the toxicity strengthened.

Project idea

Inspired by the project about AIDS, we come up with another project concerning the resistance against the virus. That is our project, the anti-phage E.coli. We use the modified promoter pRM to detect the lambda phage and use the antisense RNA and the lysis protein to defeat the phage (for more details please see the overview of our project). We are sure that there are no issues raise from our project idea for the reasons below:

The researchers:

As the project only aims at defeating the phages and protecting the bacteria for the fermentation factories, our experiments have no direct influences on the researchers.

The public:

No matter whether our project works or not, it does no harms to the public. If our project functions well, it will strengthen the resistance of the bacteria against the phages and will guarantee the quality of the fermented products.

The environment:

he engineered bacteria we design will only be used in the fermentation tanks in the factories. We have no intention to modify the organisms in the wild, so our project idea have little influence on the environment.

The strains we used

All the strains we use are the variants of the K12 E.coli. It is a version of E.coli that is thoroughly studied and found to be very safe and practical for molecular cloning research.

The researchers:

Proper safety protocols are followed relative to our biosafety level when inside this lab. This guarantees the safety of our team members when using the strains. (see our protocol)(see the overview)

The public:

Due to the principles in our lab, the bacteria used only for research will not be allowed to be taken or released out of the lab. All of our team members applied to this principle.

The environment:

The only risk exists is that if a K12 E.coli conjugates with another type of bacteria that was more harmful and confers the antibiotic resistance gene to it. The chances of this happening are small due to two important reasons: First, as was mentioned above, the bacteria used in a lab will not be allowed to be taken or released out of the lab. Second, the K12 E.coli has been adapted to the living environment of the medium artificially created in the lab. E. coli K12 has no known survival mechanisms in the wild.

The variants and their genotypes are listed below:

BL12: F-, ompT, gal dcm lon hsdSB(rB- mB-) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5])

JM109: endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB+ Δ(lac-proAB) e14- [F' traD36 proAB+ lacIq lacZΔM15] hsdR17(rK-mK+)

DH5a: F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK- mK+), λ–

Top10: F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ-

XL10gold: endA1 glnV44 recA1 thi-1 gyrA96 relA1 lac Hte Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 tetR F'[proAB lacIqZΔM15 Tn10(TetR Amy CmR)]

Phage

The phage we use is lambda phage. It is one of the most clearly studied phages and there is a set of mature, comprehensive protocols for management. We use the E.coli K12 F gal+, the double lysogen which has both the genome of the lambda phage and the genome of the defective mutant of lambda phage(λdg), to induce and extract the lambda phages.((see our protocol)

The researchers:

The lambda phage specifically infects the E.coli. It does no harm to human body. Thus the lambda phage doesn't raise any safety issue in terms of researchers.

The public:

Although some lambda phage particles may spread out of the lab by air, water or drainage system, as has mentioned above, the lambda phage cannot create any threat to people, so it doesn't raise any safety issue concerning public either.

The environment:

We think that the lambda phage we use in our lab has little passive effects on environment for three reasons below:

1. We only use the phage to test our system so we don't need too many phages. In fact, we only miniprepped 20 ml of phage lysates and when we were using them, we always diluted the lysates to low concentrations (gradient concentrations, from 10-6 to 10-18 of the origin concentration). Thus, the exact amount of phages we used is quite few.

2. We strictly abide by the protocol and the principles for keeping laboratory's safety. (see our protocols). The main methods we take to avoid the phage from spreading out of the lab are listed below:

• The super clean benches are exposed to ultraviolet rays at least 20 minutes before conducting the experiments.

• Disinfectant (main component is NaClO) is spraying thoroughly to the super clean bench before and after the experiments.

• The media (including liquid and solid medium) containing phages or lysogen are autoclaved before discarded.

3. We invite a graduate student who has rich experience in conducting phage display to be our instructor to help us miniprepping phage lysates and conducting phage testing assay.

4. There are many wild lambda phages exist in the environment. Even if there were some lambda phages spread out of the lab, the amount is so little that the phages wouldn't have ability to affect the ecology around.

Partial Reagents we use

Gelred:

Instead of using EB which is extremely carcinogenic, we use Gelred as the dye of the DNA. When using the Gelred, we always wear gloves and strictly following the protocol. What's more, when storing the Gelred, silver paper was used to cover the tube in order to avoid the Gelred from being exposed to light and decompose afterward.

Chloroform:

We use chloroform to dissolve the immature phages and the capsid protein when miniprepping the lambda phage lysates. The volume of the chloroform we used is only 1 ml. When storing the chloroform, we use water to minimize the volatilization of chloroform and a bottle to hold it, in order to avoid it from being exposed to light and decompose afterward.

Part Safety

Generally, the new biobrick parts we made this year can be divided into four types according to the parts' functions. The safety properties of these parts are discussed below:

1. parts for testing the pRM

1. plac-RBS-cI-T (BBa_K741001)
2. plac-RBS-GFP-T (BBa_K741002)
3. pRM-RBS-GFP-T (BBa_K741003)
4. plac-RBS-cI-T-pRM-RBS-GFP-T (BBa_K741004)

These parts are designed for testing the ability of the promoter pRM of being activated by the repressor CI. The abilities of these parts are only expressing the GFP and the repressor CI. These two proteins have no toxicity both to people and to the environment. Thus, we don't think this type of parts raise any safety issues.

2. parts for testing the antisense RNA of Cro and the fusion protein Crogfp

1. antisense RNA cro (BBa_K741000)
2. Fusion Protein: crogfp (BBa_K741005)
3. RBS-crogfp-T (BBa_K741006)
4. pCon(0.244)-RBS-crogfp-T (BBa_K741008)
5. pCon(0.856)-RBS-crogfp-T (BBa_K741009)
6. plac-anticro-T (BBa_K741010:)
7. plac-anticro-T-pCon(0.244)-RBS-crogfp-T (BBa_K741011)
8. plac-anticro-T-pCon(0.856)-RBS-crogfp-T (BBa_K741012)
9. plac-RBS-cI-T-pCon(0.244)-RBS-crogfp-T-pRM-anticro-T (BBa_K741017)
10. plac-RBS-cI-T-pCon(0.856)-RBS-crogfp-T-pRM-anticro-T (BBa_K741018)
11. pRM-anticro-T (BBa_K741016)

These parts are designed for testing the property of the antisense RNA of cro to repress the expression of the fusion protein crogfp. Partial of these parts are designed to make the production of anticro activated by the repressor CI. These parts can only express nontoxic proteins and mRNA, which do no harm to bacteria, people and the environment.

3. parts for testing the lysis protein

1. RBS-lysis-T (BBa_K741013)
2. pRM-RBS-lysis-T (BBa_K741014)
3. plac-RBS-cI-T-pRM-RBS-lysis-T (BBa_K741015)

These parts are designed for testing the ability of the protein lysis of killing the bacteria.

Researchers and the public:

The original organism producing lysis is E.coli and the lysis can at most killing prokaryotes but has no affects on eukaryotes. So these parts are not able to threaten our researchers and the public.

Environment:

The only risk exists is that if some of our engineered E.coli which contains these parts are released out of the lab and conjugates with other types of bacteria, these bacteria may die. If so, the balance of the ecology, especially the balance of the microbes around may be broken. The chances of this happening are small due to two important reasons: First, as was mentioned before, the bacteria used in a lab will not be allowed to be taken or released out of the lab. Second, the K12 E.coli has been adapted to the living environment of the medium artificially created in the lab. E. coli K12 has no known survival mechanisms in the wild. So our engineered bacteria hardly have chance to transfer the lysis gene to other types of bacteria.

4. Our final gene circuit

pRM-anticro-T-pRM-RBS-lysis-T (BBa_K741019)

We use this gene circuit as our final method to defeat the bacteriophages. The parts used in this gene circuit, as mentioned above, raise no safety issues.

Interview with Professors

Our institution does not have a local biosafety group or committee. Instead, we have invited some professors experienced in synthetic biology to help us assess our project. During interviews with professor Haiyan Liu from the school of life sciences we explained the project, its background, methods and process. The following is their comments concerning the biosafety and biosecurity issues relating to our project.

From Professor Haiyan Liu

The project is considered to have minimal risks.

Parts

This project does not deal with any new genetic elements. The parts used in the study like AHL, Lysis and Anticro are already existed in nature and considered to be safe in lab assembling. Should the parts be integrated to other natural organism, the engineering plasmids themselves can not rival for the natural.

Strains

The team work with E.coli K12 which is wildly studied and found to be very safe and practical for research. It is suitable for high students to conduct the experiment with E.coli K12, so as for the undergraduates of team USTC_China. With daily procedure of decontamination and sterilization within the laboratory, it is unlikely for those bacteria get out of the lab. Even if the strains should be exposed to the environment, they can not survive outside of the laboratory because of its fragile nature.

Some safety problems may arise if the engineering bacterium conjugate with another type of bacteria. However, since the team is working at the campus where no specific foreign strains(such strains may exist in hospitals) exist and proper safety precautions and procedures are well followed, the chances of the conjugation are small.

λPhage

The use of virus phages in the project is considered to be safe. Though bacteria may get infected, it is more a problem of contamination in experiment than a problem of safety. Along with the E.coli K12, λPhage is a common organism already exist in the environment. The exposition of λPhage is not gonna to raise safety issues like those chemical agents. Besides, the process of decontamination is well performed every time after the experiment.

Conclusion

The project is not introduced into human and biofabrication currently. All the work have been conducted under the essential biosafety requirements in a level 1 lab. With no modification to the genes and the parts used in the study are all exist in the open nature, the risks of the project should be minimal.

Laws and Guidelines to be considered in China

Our state government has set rules and regulations on biosafety, laboratories working on pathogenic microbes are registered and under strict surveillance. We also evaluate our experimental process according to the regulations put forth by relevant departments of P. R. China.

To get the detailed information please visit:
National Biosafety Clearing-House of China 　　Biosafety regulation on pathogenic microbes

　　

Useful Ideas for future iGEM biosafety

Our team have perceived some potential risks under the current management of registration and distribution of Biobricks. Some suggestions are proposed as follows.

When we are submitting some parts to partsregistry.org, we find that no safety informations are required when a part is registered. With no enough information about the safety of each part, future iGEM teams may have difficulty in performing their risk-assessment. Our team recommend a new function concerning the safety of each part be online at partsregistry.org, to provide the future iGEM teams with better illustration and classification of biobricks.

For the distribution issue, we noticed that the parts needed for a certain project are much fewer than the parts contained in the distribution kits. This leads to an easy access to those parts that are not referred in the project and tremendous dangers may arise afterwards. Some safety problems may occur due to the abuse of biobricks. What's more, it is difficult to conduct further investigations. To ensure that every part distributed is safely used and well recorded, we suggest that the future iGEM team should submit a requirement of biobricks before certain parts are delivered to them. Also, a confirmation should be made when a team receive its kits.