SAFETY

Safety of Staphylococcus aureus

All the experiments related to the operation of Staphylococcus aureus（S. aureus） such as examination and culturing are done in Yandu Lu’s lab in Hainan University.

Since these organisms could spread in the environment and infect human, we must consider the safety problem. Before the experiments, we have safety training. We trained to follow the safety guidelines:

Fig.1 Safety and sanitation management of Hainan University Chinese version

Fig.2 Experiment guidelines for students of Hainan University

S. aureus has harm degree of category ll needed to operate in a secondary bio-safety laboratory (BSL-2). Laboratory structural facilities, safe operating procedures, safety equipment are suitable for microorganisms with medium potential harm to people or the environment, with a level II protection level.

Safety of engineered Escherchia coli (E. coli)

In order to prevent E. coli from exposing to the environment, we have several procedures to reassure the safety for the experiments.

Before the experiments:
1.give ultraviolet radiation sterilization treatment to workbench provided an ultra-clean environment for experiments.
2.lab coats and gloves are worn to avoid touching the bacteria.

After the experiments:
1. Pour 84 disinfectant into all the culture bottles in which we grow E. coli and stay for about 2 hours, therefore killing all the harmful bacteria.
2.The waste liquids are put into waste liquid bottles which are collected into the yellow medical waste bag with contaminated culture bottles. Then the bag is disinfect by using sterilizer. Thus, bacteria are inactivated.

Fig.3 Waste liquid bottles collected into the yellow medical waste bag

Fig.4 Yellow medical waste bag contain waste liquid bottles

Fig.5 Bag disinfect by using sterilizer

Specific design of part 1

Q1: Operate the engineering bacteria.
Doing the experiments, we first give ultraviolet radiation sterilization treatment to workbench provided an ultra-clean environment for experiments. Also, lab coats and gloves are worn to avoid touching the bacteria. After experiments, ultraviolet and 84 disinfectant are used to help killing the bacteria, so no harmful bacteria left. In order to kill the bacteria, we can also use saturated steam.

Q2: Estimate the possibility of TurboID.Ecoli(BL21) poses threats to normal E. coli.
When we express TurboID in E. coli, we actually created a new so-called “species” which may compete with the current species, so that it’s our responsibility to estimate the possibility of TurboID.Ecoli(BL21) posing a competitive inhibition to normal E. coli.

During the process of TurboID’s over expressing TurboID, in order for new functions, it must experiences internal frictions, which means it will use more energy, in the forms of ATP and Biotin, than the normal E. coli. Such discovery suggests that the new species is disadvantaged during the Biologic evolution because of the limits of energy. In this way, it’s impossible for E. coli with TurboID to compete with normal E. coli since it will be shifted out by nature for certain.

Q3:Estimate the possibility of TurboID poses threats to living creatures.
First of all, TurboID usually expresses in bacteria’s periplasm, the region between the cell wall and the cytoplasm. One of the biological evolutionary advantages is that the periplasm can provide a oxydic environment, which simulate the endoplasmic reticulum in mammals so that the bacteria can fold the protein in a much easier way to form more kinds of space structures. In this way, the engineering bacteria can have more functions. On top of that, expressing in bacteria’s periplasm can help protect the bacteria so that when used in industries, it can help reduce losses and enlarge productivity. In the bacteria, TurboID is connected with a signal peptide, shown as PELB in the graph.

Specific design of part 2

Engineered bacteria part2 dethiobiotin synthase_biotin synthase

There may be potential damage to the environment due to high biotin yields, so we did conduct two procedures to prevent the damage:
1. Crispr/cas9 system will destroy the gene of Bifunctional ligase/repressor BirA which will force bacteria to be defected from other E. coli.

Fig.6 Crispr/cas9 destroy gene

2. Lac operon technique will prevent production of enzyme. Without IPTG, LacI will inhibit RNA ligase from converging with T7 promoter which will then prevent the production of mRNA. Thus, protein and enzyme production will not be available.

Fig.7 Compare pathway with and without IPTG

From these techniques, the engineered E. coli will be classified as disadvantaged and will be eliminated as organisms evolve.

Experimental safety:
Before:
1. Spray alcohol to clean visible surfaces and surroundings.
2. All equipment are sterilized in the Autoclave under 160 degrees Celsius for 2 hours.
3. Change to laboratory cloth in the dressing room.
4. Clean hands with hand sanitizer.

During:
1. Experiments with potential contamination risks were done inside the clean bench.
2. Every contagious procedure are done near the alcohol lamp.
3. Pipette tips are changed each time after liquids has been moved.
4. Volume of the pipette will be larger than the set volume of Pipette to avoid cross contamination.

After:
1. Open the UV light after using the clean bench.
2. Pour 84 bleach in all equipment used and settle it for 2 hours. Pour the liquid into waste bottle and put the wasted container into the yellow trash bag. Put both the bottle and the bag into the sterilization pot(121 degrees for 20 minutes)and seal them in a plastic bag after sterilization. Put the plastic bag into the special trash cane and carry it to the cleaner with a laboratory cart.
3. Spray alcohol to clean visible surfaces and surrounding.
4. Clean hands with hand sanitizer.

Suicide system for all the engineered E. coli

QS (quorum sensing) suicide system

This system (QS suicide system) is a system that will self-start when our engineering bacteria are lost to the nature. How does this system work? Before explaining the operation of this system, we need to know that there are lots of acyl-homomserine lactones (AHL) as it is QS signaling compounds of Gram-negative bacteria which are frequent colonizers of the nature. Also, it is noteworthy that our engineering bacteria have been artificially added with a special protein, LUXR, before doing research. In this case, whenever the engineering bacteria are lost in to the nature, the LUXR in the engineering bacteria will meet AHL as it is so common in the nature, and they will combine together to form LUXR-AHL complex. This complex will activate the cas9 in the engineering bacteria, which we added artificially. Cas9 is a 160 kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized in genetic engineering applications. Cas9 we used contains a specific sgRNA which is a specific RNA sequence that recognizes the target DNA region of interest and directs the Cas nuclease there for editing, so that they can function together to cut ATP synthase in the Staphylococcus aureus in order to disfunction Staphylococcus aureus. In addition, the sequence of sgRNA is AAAGTCGCAATTGTATGCAC. How we find the this specific sgRNA? First, we used uniprot to ascertain the specific ATP, which needs to be destroyed. The entry number of this specific ATP is P68699 in the uniprot, and the name is ATP synthase submit c. Second, we found the complete sequence of this ATP, and then use www.idtdna.com, a sgRNA design tool, to design this specific sgRNA. In this case, as we add this QS suicide system in to engineering bacteria, it can take precaution against the danger of the engineering bacteria’s leakage. The exact processes are shown on the figure below.

Fig.8 Part1 and part2 bacterial ascaped from laboratory

Fig.9 Part1 and part2 bacterial engineering status and QS suicide system silenced