Our concern: exposure of engineered Escherichia coil into the environment that have unknown result.

mazF is one of the most widely studied toxin-antitoxin systems with the most clearly identified mechanism of action which is the downstream gene of TA system mazEF(MazEF is an Escherichia coli suicide module specific for a stable toxin and a labile antitoxin. Inhibiting mazEF expression appeared to activate the module to cause cell death), encoding stable toxin protein. MazF is a ribosome-independent mRNA interfering enzyme (endonuclease) that can cut single-stranded mRNA at specific sequence sites and it is conserved in many microorganisms.

The araC promoter is based on the arabinose operon model with arabinose as the inducer and araC (arabinose operon rehulatory protein) as the surpressor.Studies have shown that arabinose can change the conformation of araC preventing it from interacting with pBad therefore inhibits it.So by controlling the concentration of arabinose, the expression of the target gene can be regulated.

(The process of araC promoter)

The araC promoter activates when it meets the the arabinose surrounding the seeds which is at its outermost layer. The mazF gene produces MazF which recognizes the ACA sequence and hydrolyzes the phosphodiester bond at the 5' or 3' end of its first A site, thereby releasing ribosomes on the cleaved mRNA and preventing protein synthesis. The abnormally encoded peptides are then released and degraded by intracellular proteases, resulting in cell death.

First surround with strain freeze-dried powder (freezed-dried so that it can only be activated when there is water).The second layer contained hydrogel that insulate the strain freeze-dried powder.The outermost layer is hydrocoagulant containing arabinose so the bacteria will be killed after the roots breaks to the outer layer.

The laboratory commonly used flammable solvents, such as ether, acetone, methanol and explosive gases and Thai drugs, such as these drugs such as improper use will occur fire, poisoning, explosion and other major accidents, so we must strengthen safety precautions, strict implementation of instrument operating procedures. For the safety of life and property and the normal development of scientific experiments, the following management system is hereby formulated, please strictly follow it.

The laboratory commonly used flammable solvents, such as ether, acetone, methanol and explosive gases and Thai drugs, such as these drugs such as improper use will occur fire, poisoning, explosion and other major accidents, so we must strengthen safety precautions, strict implementation of instrument operating procedures.

For the safety of life and property and the normal development of scientific experiments, the following management system is hereby formulated, please strictly follow it.(These guidelines do not coverall hazards and possible dangerous scenarios and do not excuse the student from being alert to other hazards in the lab that are not covered by this guide.)

Each student must always bear in mind that he or she is not alone in the lab, and that their actions may have some consequences on other people around them.

The goals of these guidelines are to:

a. To teach students about formal and safe behavior

b. Warn about the dangers while working in a lab

c. Introduce students to the safety infrastructure and equipment that are at their disposal in lab.

We briefly describe how to deal with dangerous scenarios and how to act in cases of injury in the lab.

The guidelines are aimed at all students in the labs of the research institute of Tsinghua University in Shenzhen

All students must read the safety instructions and sign the attached confirmation slip. Without doing so, the student will not be allowed into the lab.

Student must use personal protective/safety equipment (PPE) at all times, including:

1. Safety glasses/goggles.

2. Long lab coat, buttoned and with long sleeves

3. Protective gloves in labs

4. Closed shoes, preferably leather or plastic (not fabric). Sandals (slippers),

flip-flops as well as high heels are strictly forbidden in the lab.

5. Additional safety gear (safety mask, apron, or heat resistant gloves etc.) is

required in special experiments according to the instructor’s guidelines.

6. Hair must be adequately tied back (and preferably, covered) during work

in the lab.

7. Hands must be washed frequently.

1. When you finish working in the lab, disconnect all systems and equipment from gas, water, electricity, according to your instructors’ guidelines.

2. Return all chemicals and moveable equipment to their place, as soon as you are done using them.

3. Verify that there are no chemicals left on the working space or on the floor. Clean all glassware and dirty surfaces according to your instructors’ instructions.

4. Decontaminate the work space and your personal safety gear, according to your instructors’ guidelines.

5. Wash your hands with soap and water when you are done working.

6. In case of a chemical liquid or powder spill, use the designated equipment to treat it, following the guidelines given by your instructor. Remember to protect yourself with safety gear before using any neutralization solution or absorption chemical.

7. Handle chemical waste according to your instructors’ guidelines, i.e., neutralize acidic/basic waste; pour organic solvents into appropriate waste containers.

8. Systems that are left to work continuously and do not require the presence of the user. It should be only receiving direct authorization from the lab instructor (preferably written) and will be operated only according to the instructors' guidelines.

1. Be familiar with the equipment and its instructions

2. Locate the emergency switch and learn how to use it. In case of electrocution or any

malfunction, treat any electrical wire as though it is “alive” .

3. Do not modify or alter any electrical machine or device or perform any modifications

to the lab electrical system.

4. Do not use a device, plug, or any part of a system if it is broken or defective.

5. Never touch an electrical instrument or device, wire, or plug with wet hands.

6. If a flammable liquid has been spilled on or around an electrical device, immediately remove people from the surroundings and alert your instructor.

7. When you finish working, disconnect all electrical devices, according to your instructor’s guidelines.

Stay when the infrastructure is still working

Beware of electricity

Beware of high heat

a. Keep a safe distance while heating any substance

b. Wear thick gloves in case of burns

c. Do the experiment in the fume hood

d. Remember that vapors of flammable liquids can spread by heat source

Keeping a safe distance

Burns

(1) Minor burn – the skin is intact – immerse skin in ice-bath or cold water

(2) Serious burn – the area of the burn is large or the skin is broken – remove clothing from the area by cutting off and go immediately to the hospital.

a. Remember that dilution of acids and bases can be highly exothermic. Therefore,

never pour water into an acid. Dilute them by pouring the concentrated acid or

base solutions into water, while constantly stirring and cooling.

b. Protect your eyes with safety glasses and your skin with gloves and appropriate

clothing (like an apron) when working with concentrated acid or base solutions.

Follow the manufacturer and your instructors’ guidelines.

c. Neutralize acidic and basic waste solutions before discarding, according to your

instructors’ guidelines.

In case of contact with a chemical or injury, the type of first aid depends on the specific case:

a. Chemical in the eye

Wash the eye thoroughly at the eyewash stand for at least 15 minutes. First aid will be administered on the spot by one of the instructors or by peers/friends.

(1) Acid in the eye - Wash immediately and thoroughly with water, using the eyewash stand.

(Try to keep your eyes open) It is important for the wounded person to see a doctor.

(2) Base in the eye - Same as with acid, but also wash with 1% boric acid solution. It is important for the wounded person to see a doctor.

Detailed process of using the eyewash

Eyewash stand equipped in every room

First aid kit in every room in case small emergency(severe injury needs to go to the hospital)

Frequent detection in the equipment in the lab

##Emergency procedure in case of fire

a. Using the extinguisher – allowed only to those who have been instructed on how

to use the extinguisher. Using a water hose to put out a fire is forbidden unless

instructed specifically by an authorized personnel.

b. Clothes on fire –

(1)Prevent the burning person from running around and aggravating the fire.

(2)Extinguish the fire by wrapping the person with a thick blanket or lab coat.

If possible,use an emergency shower or strong current of water.

(3)Do not extinguish the fire with an extinguisher.

##Disposal of waste

There are three basic routes of disposal of laboratory chemical waste:

Sanitary sewer or trash disposal of non-hazardous materials;

Acid-base neutralization, followed by sewer disposal. (Note:Any other type of treatment must be part of an experimental procedure to be considered legal collection for both laboratory and non-laboratory operations)