Team:TJUSLS China/Safety

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SAFETY

Introduction

We promise none of the organisms we use pose a risk to the safety and health of team members, and no organism can cause infection or adverse health effects or symptoms. The detailed safety measures can be found below.

Prior to Experiment

Safety form

When coming up with our project, we regard the safety assessment as a necessity. We completed the safety form and started up with constructing our parts of PETase genes after we received the approval from safety committee.

Biosafety Training

1.Course

Before we were allowed to enter the laboratory, we were given a course called Biosafety and bioethics. The teachers informed us of all the potential risks and dangers associated with the use of different chemicals, techniques, and equipment, as well as how we could minimize these risks. Following this, we were given a laboratory induction by the teacher who demonstrated the safest and most efficient ways to carry out the protocols we would require.

2.Test

Members of the lab group have passed the bio-safety training and testing required by laboratory assistants and engineers, and only after passing the tests can we conduct experiments. The standard of passing this test is 90%.

Safe Labwork

Organisms and parts

1. Risks of parts

During our experiment, no harm to humans and the environment due to PETase has been found yet.

2. Kind of bacteria

Our project poses little risk to the students, as for the only organism used in our project is a commonly used lab-strain of Escherichia coli, which is designated as a biosafety level 1 organism. E.coli DH5alpha was used for molecular cloning to obtain recombinant plasmids and E.coli BL21(DE3) was used for protein expression and purification to obtain target proteins.

3.Gene Transfer Prevention

Bacteria have three methods by which DNA may be transferred from one cell to another: transformation, transduction and conjugation. Plasmids and conjugative transposons transfer from one cell to another by mechanisms that involve replication. Transposons, gene cassettes and ISCR-mediated gene transfer between sites on the same or on different DNA molecules require some form of recombination. Plasmids accumulate antibiotic resistance genes as a consequence of the activities of at least these three recombination systems. Since the detailed mechanism of these recombination systems of gene transfer remains unclear, the only thing we have to do—the most important thing at the same time—is to package the genes in order to prevent its spread inside or outside the lab. To prevent antibiotic-resistant genes being released into the environment, all of the operations related to living bacteria carried with them are in the clean bench. All of the bacterial liquid is collected specially in a waste bucket with high concentration of NaOH to kill phage and change the structure of DNA. Used tubes and plates are all autoclaved.

Facilities and operations

1.Facilities

This year our project employed the use of laboratories classified as biosafety level (BSL) 1. The building of laboratory includes the location of emergency exits, emergency ventilation system,  first aid kit and so on. Emergency eye washer is situated outside of our lab, together with aid supplement and fire extinguishers.

2.Operations

Aseptic techniques are always used when handling live culture or mixing some buffer. Super clean bench is used when doing any operations about living bacteria, for example, taking monoclones from colony in plate. Gloves and lab coats are required to wear as soon as we step into the laboratory. Our dressing seriously follow BSL-1 laboratory standard. In EB contaminated area, agarose gel analysis of DNA is performed with Ethidium Bromide stain and always imaged safely in a gel imager. We are required to wear double gloves when adding EB into agarose gel. And all of the facilities such as measuring cylinder in this contaminated area cannot be taken to other benches.

Safety Rules

1.Supervision

We never work in the lab alone and there have been a minimum of two or more students from the team at all times. In addition to this, a responsible from our supervisors research group have been available and nearby at all times.

2. Laboratory Safety Rules

Our lab follows China National Institute of Pathogenic Microorganisms Biosafety Management and Tianjin University Laboratory Safety Rules.

Figure 1. Student Handbook for Laboratory Safety

Subsequent to Experiment

Waste disposal

All of the waste would be divided into relevant categories and collect each of them separately. There are some mini dustbins on the table collecting pipettes and microtube, and by the end of a day’s experiment we will pull them out into dust bin in the lab. And the person on cleaning duty that day is responsible for changing the yellow garbage bag and taking the full bag to school’s collection site.

Useless bacterial liquid are pulled in a bucket together with high concentration NaOH. This bucket is experimented with two lids, meeting our country’s standard.

Hygiene

As mentioned, our lab has different person on cleaning duty everyday in turn and all of our team members are on the list. Not only should this one deal with garbage, but he is also required to wipe and mop the ground, clean the public table, check facilites and note all of his tasks in the chart.

70% ethanol is used for disinfection anytime.

Everyone has formed the habit of washing hands with antibacterial hand sanitizer at the exit of labtorary when finishing experiments.

Reference

[1]Regulations on Biosafety Management of Pathogenic Microorganism Laboratories. http://www.gov.cn/zwgk/2005-05/23/content_256.htm

[2]Measures for the Biosafety Environmental Management of Pathogenic Microorganism Laboratories.2006.3.08.http://www.mee.gov.cn/gkml/zj/jl/200910/t20091022_171837.htm

[3] iGEM team TJUSLS_China2019. Safety. https://2019.igem.org/Team:TJUSLS_China/Safety

[4] Bennett, P.M., 2008. Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria. Br. J. Pharmacol. 153 (S1), 347–357.