General lab safety

Our laboratory upholds a set of biological safety protocols including biosafety management and waste categorization. We have completed safety training sessions organized by the Laboratory and Equipment Management Department of Zhejiang University. We also learned how to be careful with genetically engineered bacteria and components, followed by the biosecurity guidelines provided by Zhejiang University, including the Zhejiang University Laboratory Safety Management Measures and the Zhejiang University Laboratory Safety Manual.

Those who have not passed the laboratory safety test are not allowed to enter the laboratory to conduct experiments. Members must abide by the rules of the laboratory and strictly enforce the safety procedures. Members must be familiar with the various sources of hazards in the laboratory and master the protective measures and emergency disposal methods. Members must wear the appropriate personal protective equipment when entering the laboratory. There are no items or behaviors in the laboratory that are not related to the experiment. A safety duty system is established in the laboratory. At the end of each day, personnel leaving the laboratory should check water and electricity safety and keep records.

LqhIT2 toxin safety

LqhIT2 has been studied since the 1990s. Many experiments and dozens of papers have confirmed that it is an insect-specific toxin. One of the most convincing experiments was done by Zlotkin et al. in 1991. They injected purified LqhIT2 into live mice, and it showed no toxicity. This experiment was repeated by Herrmann et al. in 1995, and their results are consistent.

Cohen et al. reconfirmed that LhqIT2 alone is not effective on mammals in 2007. They found that LhqIT2 is effective on mammals only when the voltage-gated sodium channel is excited before toxin application by either a long depolarizing prepulse (artificial manipulation) or by modulation with an alpha-toxin (presents in complete scorpion venom, but not in our product).

The most recent research on LqhIT2 was published by Zhu et al in 2023. They analyzed the molecular structure of LqhIT2 and compared it with other toxins. They concluded that the channel residue and cavity shape of LqhIT2 determine its species selectivity of insects.

We will not try to extract or purify the toxin. So, we will not be in contact with the toxin directly. We will only insert the LqhIT2 gene downstream of an insect hemolymph-specific promoter (Pmcl1). So LqhIT2 will only be synthesized by our fungi in the insect bodies. Even if LqhIT2 leaked from insect corpses, as a 61-amino acid peptide, it will be degraded rapidly in the environment by microorganisms. Besides, plants are not capable of absorbing intact peptides longer than five amino acids (Tegeder et al., 2010). As a result, it is not possible that the toxin LqhIT2 will enter the crops and be transferred to the human side.

Genes can mutate, and we wanted to be sure that mutated LqhIT2 would not become toxic for humans. So, we ran Protein Blast on NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins). Results showed that genes share a similar sequence with LqhIT2 all encode for insect-specific toxins, which showed the safety of LqhIT2.

Gene	Query Cover	E value	Per. Ident	Accession	Toxicity
LqqIT2	98%	4.00E-33	88.52%	P19855.2	Insect-specific
LqhIT5	98%	9.00E-23	73.77%	P81240.1	Insect-specific
BotIT4	98%	8.00E-34	95.08%	P55903.1	Insect-specific
BotIT5	98%	3.00E-33	93.44%	P55904.1	Insect-specific
BotIT6	98%	1.00E-18	63.93%	P59864.1	Insect-specific
BjIT2	98%	1.00E-28	78.69%	P24336.1	Insect-specific
BmKITa	98%	4.00E-29	81.97%	Q9XY87.1	Insect-specific
BmKITb	98%	3.00E-28	80.33%	Q95WX6.1	Insect-specific
BmKITc	98%	2.00E-24	75.41%	Q9Y1U3.2	Insect-specific
BmKAEP	98%	2.00E-29	80.33%	P15228.2	Insect-specific
BmKAEP2	98%	1.00E-28	81.97%	Q86M31.1	Insect-specific
BmKIT2	98%	1.00E-28	81.97%	P68727.1	Insect-specific
BmKIT3	98%	8.00E-29	83.61%	Q17231.2	Insect-specific

(Moskowitz et al., 1998; Li et al., 2000; Ali et al., 2001; Goudet al., 2002; Peng et al., 2002)

Fungus safety

Metarhizium anisolpliae is native to China and has been commercialized and widely used in China and around the world. It does infect mammals, so it is a safe species for humans.

However, as a fungus, M. anisolpliae produces spores and spores are prone to accidental leakage. We take measures to prevent spore spread. We keep our fungi in Petri dishes (solid media) or capped laboratory bottles (liquid media). All the containers are carefully sealed by Parafilm. These Petri dishes and bottles are kept in a constant temperature incubator that is only used for storing our fungi. All the containers are autoclaved after use to kill the fungi. UV lamps and ethanol wipes are used to sterilize the incubator after use. Taking samples out of the lab is strictly forbidden. The containers are only allowed to be opened in biosafety cabinets. When working on our fungi, all the members are required to wear nitrile gloves and N95 masks. All the samples are well-labeled. After each experiment, all the disposables  (gloves, pipette tips, etc.) are autoclaved before disposal. UV lamps in the biosafety cabinets will be turned on for at least 40 minutes after use.

We also try to limit unneeded spore formation. Fungi tend to undergo vegetative growth in suitable environments (sufficient nutrition, low UV, suitable temperature, low disturbance) and form spores otherwise. When we do not need spores, we grow our fungi on full PDA/PDB media at 30 ℃ degrees, and no spore formation can be observed for at least two weeks.

Besides, we include a suicide switch in our engineered fungal pesticide. Our fungi will contain a photosensor that leads to the inactivation of new-forming spores under the sunlight. This will prevent its unintended spread into the environment.

Insect safety

Our goal is to improve a fungal biopesticide. So, we need to test its effectiveness and efficiency on some insects. We use Galleria mellonella larvae to do the test.

We use G. mellonella only to test our pesticides, so we will not release them into the environment. To prevent the larvae from escaping, we keep them in sealed boxes and store them in the refrigerator before use. When used in the experiment, the larvae and our pesticide are kept in Petri dishes and placed in a constant temperature incubator. The larvae we use are big caterpillars (3/4 inch long) that are easy to spot and have very limited mobility. So we believe the larvae of G. mellonella cannot escape from their containers.

Unlike chubby caterpillars, the adults of G. mellonella are moths that are more likely to escape. So we check all the larvae every day to see if they pupate. Any pupa will be killed instantly to prevent eclosion.

References