Team:Tsinghua/Safety

We have always attached great importance to the bio-safety in our experimental progress. And we have been fully implement experimental safety guidelines to ensure the safety of our teammates and to keep a healthy environment. The strains of Escherichia coli we used are harmless, which are widely used in all kinds of laboratories around the world.We answered the questions about safety on the Safety Page from iGEM(https://2012.igem.org/Safety) as following:

Q1

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

A1：Our project is to use E.coli to transport and amplify signals in the media and no serious safety problems will be caused. Firstly, the engineering strains of E.coli are harmless. Secondly, we use ampicillin to select our desired strains, thus only strains with our functional genes can survive. Thirdly, in case of the possible problems, we design a turn-off module in our project to stop the functional part and also add a suicide gene to kill all the bacteria. So we are quite sure that our project will not raise any safety issues in terms of the above problems.

Q2

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?

A2: No, the parts we used/designed this year do not raise potential

safety issues.

Q3

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?

A3: We do have a local biosafety group in the microbiology lab, which
had trained our team of distinct aspects before starting lab work,
including lab safety, waste treatment, potential safety issues in the
design and etc. During the first few weeks of experiments, at least
one member of the group would supervise our operation and ensured that
no research or environment safety problems would occur. Also, we had
some supervisors/professors from School of Life Sciences, Tsinghua
University to examine our lab work. They scrutinized by raising
questions to our member directly as well as by watching us carrying
out certain procedures. The local biosafety group praised our team for
the caution on safety issues, and highly approved our design of parts
that may contribute to improve bioengineering.

Q4

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?

A4: We believe that the most incentive way is to establish a corresponding award. This award can be used to reward those teams who build some good bio-security parts or modules. At the same time, to improve the situation, the experience of previous years and some very practical biosafety precautions can be summed up, which can be distributed to the participating teams each year. Also, the heritage on the safe operation within the same school can also be encouraged. Some practical and important safety details can even be published as operation Manual, which will facilitate other teams learning.

Our design for biosafety

At first, the model bacteria DH5 alpha and BL21(DE3) that we used in our project are harmless. All of the virulence genes have been knocked out. As a result, the bacteria themselves are safe to our researchers and environment.
Secondly, our part is built in the plasmid which contains antibiotic coding sequence. When the cells are cultured in the LB medium, we will add antibiotic such as ampicillin or kanamycin into the medium. In this way, only the cells that contain our functional part can survive. Once the plasmids are lost, the cells will die.
Thirdly we design the “turn-off” part which can shut down the expression and the working state of our project just like one power switch of our project. We use the invertase FimE to accomplish this job.

Introduction of FimE: 
 FimE is one of the invertase that can recognize and then bind with the specific sequence of DNA. We can call the IRL which is the recognition site on the left of target gene and IRR which is on the right. Our idea is to use the nature of FimE to invert the promoter, thus leading to the shut-down of expression, even to the suicide of bacteria. This kind of reverse is inreversible so that we can use this enzyme to make sure that we can stop our part from expression.

Stop working!
Since FimE does exist in the genome of E.coli, we first have
to knock this element out. The next step is to insert FimE into the
plasmid, downstream of T7 promoter and lac operator, as a part of plasmid backbone. The expression of FimE can be induced by IPTG.
We added the following part upstream of every functional part of our experiment, which functions as a switch to shut down its expression, and carrying out other tasks to ensure biosafety. Once FimE is expressed by adding IPTG, it will bind to its specific recognition sequence IRL & IRR, inverting this part. As a result, the promoter is turned to the opposite direction and doesn’t make any sense because of the terminator.

Good-bye, E.coli!
In order to prevent some potential problems, we design a module which can directly lead to the suicide of bacteria. We use lysozyme as our suicide protein, and put the gene on the upstream of a promoter which has FimE recognition parts on both sides. Since we already have FimE in our designs, which can be directly induced by IPTG, we only need to add IPTG to start the suicide program, and the FimE will bind to the recognition parts to inverse the promoter and express lysozyme. In this way, we can directly kill all the bacteria that may raise safety issues.