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.
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.
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.
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.
|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.
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
|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