Safety Exam:

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Participants of our wet lab have learned about P2 lab safety protocols and accomplished the P2 safety exam, which is mandatory for all members working in P2 labs, provided to us by the School of Life Science in SUSTech.

Risk:

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Organisms we use in our project:

Escherichia coli WM6026, T-fast, pir1 (BSL-1)

Vibrio cholerae V52 rhh (BSL-2)

Pseudomonas aeruginosa PAO1 DEUC (BSL-2)

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Risk of the Organism:

Laboratory bacteria can escape in the laboratory for a variety of reasons, especially if the subsequent cleanup is mishandled.

Mutation may occur in the case of improper preservation.

In the case of experimental mixing, it is possible to produce toxic variation.

It may be mixed with other experimental substances to cause mutations, etc.

V. cholerae and P. aeruginosa are pathogens, so upon exposure, they may cause disease.

Management:

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Management of Lab:

Our experiments involve using BSL-2 risk-level organisms, to ensure our safety, all safety measures of the BSL-2 laboratory are strictly kept, including:

• We are equipped with lab coats, gloves, eye protection, and face shields when conducting experiments.
• Procedures with the possibility of aerosols or splashes are done in the biosafety cabinet.
• All members of the experiment group have learned about the safety measures of the BSL2 lab and taken exams about the safety protocols of the BSL2 lab to gain access to the lab.
• The experiments we conduct are supervised by our professor, who is experienced in using V. cholerae and P. aeruginosa as experiment material.

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Lab equipment:

Here we present some of the safety equipment of our lab.

Figure 2. Lab coats and gloves of our lab.

Figure 3. Autoclave of our lab.

Figure 4. Biosafety cabinet of our lab.

Figure 5. Bacteria storage refrigerator.

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Management of Organism:

Not only do we hope to make our experiments safer, but we also want to provide a safe and efficient bacterial strain selection for other teams using T6SS in the future. Therefore, the strains we use in our projects are mostly attenuated, and the attenuation modifications done on these strains are as follows:

The following genes are knocked out from Vibrio cholerae V52 in the rhh strain:

• rtxA toxin, causing severe β-hemolysis
• hlyA component of Type I Secretion System
• hapA necessary for Vibrio cholerae to move through the mucosal layer
• vasX t6ss effector targeting eukaryotic amoeba and bacterial cells
• tseL promoting bacterial internalization into host cells
• tesH cell wall lysis effector

The following genes are knocked out from Pseudomonas aeruginosa PAO1 in the DEUC strain:

• tse1-8 effectors of t6ss, toxins

We also knock out the following genes to ensure its safety:

• exoS & exoT t3ss exoenzyme, induces host cell apoptosis
• vfr cAMP-binding transcriptional regulator that controls the production of multiple virulence factors

The rationale for Using These Strains and Knockout:

• For Vibrio Cholerae, V52 is from the Vibrio Cholerae O37 serogroup, compared to strains from O1 and O139 serogroups, it does not carry the main virulence factors (cholera toxin and toxin-coregulated pilus) which causes severe diarrhea and dehydration 2. We utilize this strain because it can constitutively produce T6SS and is commonly used in research related to t6ss 3. Science literature has suggested that hapA, hlyA, and rtxA contribute to most of the toxicity of non-O1, and non-O139 Vibrio cholerae (NOVC) 4. An article published also provides an assay showing the knockout of hapA, hlyA, and rtxA reduces cell death rate. We use a substrain of V52 called rhh as most of its virulence genes including rtxA, hlyA, hapA, vasX, tseL, and tesH are knocked out, in which vasX, tseL and tseH are the original effecters of T6SS.


Figure 6. A LDH-based cytotoxicity assays done on HeLa cell line proves that knocking out of hapA, hlyA, and rtxA can significantly reduce cytotoxicity [4].

• For Pseudomonas aeruginosa, PAO1 is also a commonly used strain in T6SS research. The strain we choose, DEUC, is a substrain of PAO1 engineered specifically for T6SS expression. However, it still holds some virulence genes. So, we follow a synthetic biology research article published in the National Science Review to attenuate this stain 6. The researchers of the article also did a survival rate test on the knockout strain to test for the result of knocking out exoS and exoT. The graph provided in the article proves that the knockout of exoST can increase the survival rate of infected mice. Therefore, in our experiment, we have knocked out exoS, exoT and vfr for attenuation, and compared to normal Pseudomonas aeruginosa PAO1, it also lacks tse1~8 which are the original effectors of T6SS.


Figure 7. Survival rate assays done on mice prove that knockout of exoS & exoT can remarkably alleviate the death rate of infected mice [5].