How to help the E.Coli resist heat?

There is a protein family called HSP. They can help cells to fix the damage caused by heat by restoring the protein back to normal condition. We implant HSP from hot spring ecosystem into E.coli. As a result, E.coli with our HSP can keep their producing ability, ignoring the hot environment. It can be seen in our customize thermotolerance system.

How to control the cell density?

Maybe many people think bacteria are selfish creatures, but that is not totally true. MazEF, is a widely used system among wide bacteria. With this system, many bacteria will sacrifice themselves, and leave nutrition to the left ones when facing threats from dangerous environment, such as heat, lack of food and so on. MazF , which has RNA cleavage activity, is a kind of toxin. MazE, on the other hand, is the antidote of MazF. When MazE combine with MazF, the cell will not be harmed. But once we inhibit the production of MazE, the cell step into programmed cell death(PCD). In our design, we can control the expression of antidote to control the cell density. That is our third device of intelligent quorum regulating system.

What means “when it is properly to do so”? And how to fulfill that?

Every new gene is a burden to E.coli. HSPs expressed under a cool condition or extra expressed HSPs are useless, which will disturb normal production progress. We must make control when and how many HSP should be expressed.

We use RNA thermometer as our switch. This kind of RNA has a special stem circle structure, which is opened only when the temperature reach the set status. Then we can make HSP expressed in a hierarchy way.

Similarly, the problem of “when” and “how many” also appears on the control of cell density.

We must ensure that PCD happens when the cell density is high, and the extent must be gentle. Otherwise the cell density might decrease directly and cause negative affect on production. we use quorum sensing device to settle this problem.

Organism & Experiment safety

The organisms used in our experiment conclude:

• Escherichia coli BL21(DE3)
• Escherichia coli K12

Although all of the organisms are in line with biosafety level 1 (BSL), we have treated it as a potential affection source and created following rules to minimize the danger:

• Wearing appropriate protective equipment like gloves and lab coats.
• Don’t eat any food or store it in the lab.
• Avoid wearing shorts or shoes that make your legs or toes exposed.
• Washing hands before leaving the laboratory.

Safety training

There is no Biosafety Committee at our university, only a bio-safety officer. Nevertheless, we have iGEM instructors, Dr. Chun Li and Dr. Shuyuan Guo, who trained us about the project progress regularly and is responsible for the bio-safety of our project. The general rule at our university is that every student has to take part in specific safety training before their first experiment. Further, once a year, every member of the lab is required to take a refreshment course in lab safety, bio safety, fire safety, waste management, handling gas cylinders and working with hazardous materials. All of these treatments ensure our lab safety.

Public & Environment safety

The model organisms used in our project are non-virulent strains which are commonly used in microbiology laboratories. The organism has been provided various resistance of antibiotics such as chloramphenicol, ampicillin and kanamycin. Without the circumstance which contained specific antibiotic, our organism has no advantage in the competition with the wild one. Besides, although we have improve its resistance of higher temperature(42~45℃), it can still be denatured via heat. In a word, it can work well in laboratory environment and will be eliminated through competition when exposing in the wild.