Team:Jiangnan China/Safety

Synthetic biology is based on biology, chemistry, physics, computing, engineering and other multi-disciplinary cross, for organisms to redesign in the form of engineering and even from scratch, to overcome the limitations of natural evolution, create ability beyond the natural life of synthetic biology. With the development of synthetic biology technology, the application of synthetic biology is becoming more and more widely. How to protect the biological safety of synthetic biology has become a very important and key problem to be solved. Our experiments are in strict accordance with the laboratory biosafety code and protection regulations. Our lab is level 1 with low risk. And our work area is open bench. Although we all have experimental experience, we still have carried out corresponding experimental safety education and skills training to ensure the safe operation of the experiments.

Training:

Our PI Guocheng Du will be responsible for the safety of our laboratory. For the L.lactis used in the project, our Secondary PI professor Juan Zhang has long-term research experience on it. Therefore, we can ensure the safety of the organisms in the project. In addition, the advicors will stay with us during the entire experiment to ensure the safety of our experiments.
All of our experimenters have already received safety training. Training content including biological safety related laws, regulations, measures, standards, our laboratory biological safety manual, biological safety management system, emergency plan, emergency reporting and disposal process, biological safety risk assessment, biological safety operation specification, equipment use, maintenance, proper use of personal protective equipment, bacteria strains and samples of the collection, transportation, storage, use, destruction, laboratory of disinfection and sterilization, infectious waste disposal, first aid, etc.

Protection:

    1. Safety Clothing:
    All lab members wear lab coats, nitrile gloves, closed-toe shoes, and goggles to ensure personal protection from any harmful chemicals and microbes.

    2. Equipment:
    We used a UV lamp to visualise and cut agarose gels. We had to ensure we had no exposed skin on our hands/arms and had to wear a visor to protect our faces and eyes. Additionally, if any accidental emergency were to happen, all members of the team knew where safety equipments such as fire extinguishers, emergency shut off valves, eye wash station, etc., were located and how to use them. Furthermore, to prevent confusion, all laboratory equipment, such as test tubes, and bottles, had labels with proper identification to ensure that accidental usage of the reagents and other media was prevented.

    3. Operational safety:
    When the experiments are operated in Clean Benches, we light the alcohol lamp in the first place, and then disinfect gloves with alcohol, preventing hands from burning. Gloves are replaced and hands are washed immediately after using ethidium bromide or any of the harmful solutions. There are taped off, designated areas for working with ethidium bromide. These areas are cleaned before and after work and are the only areas the solution may be touched.

Material:

-Bacteria

We will use E.coli MC1061, Lactococcus lactis NZ3900, Lactococcus lactis NZ9000 and its mutant strain L. lactis WH101. No viruses will be used. Our chassis organism is Lactococcus lactis. The strains (Lactococcus lactics NZ3900 and NZ9000) involved in this experiment do not produce any toxic substances or carry resistance marker genes. Lactococcus lactis do not multiply in the form of spores. All the genetic recombination operations are based on homologous gene recombination, therefore they are relatively safe. Even if they accidentally escape the lab, they will not cause large-scale proliferation or have adverse effects on the surrounding environment.

Strain Name	Source	Characteristic
Lactococcus lactis NZ3900	Jiangnan University	Standard strain foe food grade selection based on the ability to grow on lactose. It is a progeny of NZ3000, a strain in which the lactose operon, that is generally present on plasmids, has been integrated into the chromosome and the lacF gene was deleted. Deletion of lacF gene makes this strain unable to grow on lactose unless lacF is provided on plasmid.
Lactococcus lactis NZ9000	Jiangnan University	Standard host strain for nisin regulated gene expression (NICE). The strain contains the regulatory genes nisR and nisK integrated into the pepN gene (broad range amino peptidese).
Lactococcus lactis WH101	Jiangnan University	A mutant strain of L.lactis NZ9000, and has anti-acid ability.
E.coli MC1061	Jiangnan University	A standard strain of E.coli and it can provide the gfp gene.

-Other materials:

In the experiment, we sometimes use some chemicals that are toxic to the body. In this regard, we will strictly follow the laboratory's requirements and conduct experiments in a fixed area to ensure safety. For example, stain was added during agarose gel electrophoresis. Although the new stain sYBR Green was less toxic than ethyl bromide, we followed the highly toxic protocol to wear two layers of rubber gloves during operation to avoid direct contact with gel and

-Plasmids

Plasmids	Source	Characteristic
pNZ8149	Lactococcus lactis NZ3900	Broad-host-range vector; lacF for food grade selection for growth on lactose; nisA promoter followed by a NcoⅠsite for translational fusions at the ATG. Contains a terminator after the MCS. Sequence adaption for cloning into NcoⅠcan result in a change in the second amino acid of a protein.

-Medium

M17
Eliker