Safety

We have established a set of laboratory safety regulations to guarantee the safe of team members in laboratory operations. These regulations include the subsequent guidelines:

Aspects	Rules
Personal Safety	1.Wear lab coats, gloves, goggles and close shoes when entering the lab. 2.Pay attention to personal hygiene: wash your hands frequently, avoid touching your eyes, nose and mouth, and thoroughly disinfect hands before leaving the lab. 3.Keep personal belongings properly and do not bring anything unrelated to the experiment in the laboratory. 4.Don’t smoke, eat, makeup or do other things unrelated to the experiment in the laboratory.
Equipment Use	1. be familiar with the uses and use methods of laboratory equipment. 2.Strictly comply with the Usage specification of biosafety cabinets, centrifuges, ultrasonic machines and other equipment that may produce aerosols to avoid aerosol leakage or overflow accidents.
Biosafety	The proper use and management of biological materials, such as bacteria, viruses, cells, blood, secretions, etc. should be classified, labelled, stored and transported according to different risk levels, avoiding cross-contamination or accidental exposure, and be inactivated or destroyed in a timely manner after use.
Chemical safety	Handle flammable, explosive, toxic, corrosive or radioactive chemicals with care, prepare, use and store them according to prescribed methods and conditions, and avoid contact with skin or eyes.
Fire Safety	1.Keep electricity safety: do not pull the random wire or socket and use electrical equipment in the place of damp or flammable and explosive materials. 2.Keep fire safety: do not use open flames or electric heating appliances on the experimental bench.
Safety Management	1.Keep the laboratory clean and ventilated, clean the stains and debris in time, regularly clean and disinfect the laboratory utensils and equipment, and dispose of laboratory waste according to the prescribed rules and routes. 2.Actively participate in laboratory safety management and supervision and report and rectify problems in time.
Training and Test	1.Attend safety training and pass the exam. 2. Learn emergency plans and report accidents proactively.

To guarantee that all team members possess sufficient knowledge of the laboratory safety regulations and safe usage of facilities to adhere to these principles at any time during the study, we have held the laboratory safety training session.

Afterwards, we conducted a laboratory safety examination to assess the team members' completion of the training.

• examination content: We selected 80 questions from the question bank as the laboratory safety examination paper, according to the different questions, the score is different. The total possible score is 100. (Find the laboratory safety exam question bank here.)
• examination requirement: we stipulate that we must complete the assessment content within a limited time and reach 90 points or more.
• The results of each member of the wet-lab group who have passed the laboratory safety examination are as follows:

Fig.1. wet-lab group members' laboratory safety test scores

We have made the relevant safety knowledge of laboratory toxic reagents into posters and displayed throughout our laboratory, so as to better remind team members to pay attention to daily laboratory protection and maintain laboratory safety.

Bt toxin Cry3A-like protein: Eco-friendly and Safe

Bt (Bacillus thuringiensis) is widely recognized as a safe and environmentally benign insecticide. The conditions for determining the effectiveness of Bt insecticidal proteins in killing insects are as follows:

Receptor Presence: For Bt insecticidal proteins to be effective, receptors specific to these proteins must be present in the midgut cells of the target pests.

Protease Resistance: Bt insecticidal proteins should resist rapid digestion by the proteases found in the midgut of these pests.

Impact of Bt Proteins on Human Health

The Cry3A-like protein modified by our team could only function at pH 5-7 with the presence of aminopeptidase nitrogen and calreticulin analog receptors. Since there is no receptor for Bt insecticidal proteins in the human midgut, and our engineered bacteria and secreted insecticidal proteins cannot escape degradation by human gastric digestive enzymes(Extoxnet, 1996)or strong acid stress, therefore it is not harmful to human health.

Impact of Bt Proteins on the Environment

Bt protein is moderately present in soil and is in the stable category. Its immobility prevents it from seeping into groundwater. (Xiaoyi, Wang et al.,2016)

Impact of Bt Proteins on Animals and Insects

The Cry3A-like protein has demonstrated control over pests such as fire ants, carpenter ants, Argentine ants, and Pharaoh ants(Bullaet al.,2003) It has also proven 2 non-toxic to dogs, guinea pigs, rats, fish, frogs, fire lizards, birds, as well as beneficial and predatory insects like bees and ladybugs(Extoxnet, 1996)

CPTI: Eco-friendly and Safe

CPTI, a member of the Bowman-Birk protein family, has demonstrated its potential benefits in the prevention and adjuvant treatment of cancer through numerous medical studies, with no reported harm to humans. Anti-insect spectrum tests show that CPTI inhibits almost all tested major agricultural pests. It targets the digestion process of insects that rely on serine protease as their digestive enzyme while remaining non-harmful to beneficial insects like bees.(Xuhong-linet al.,2008)

Why Establish a Multi-Insect Resistance Mechanism?

It has been shown that pests are easy to developing resitantance to a single gene. Currently, establish a multiple insect resistance mechanisms is an important and feasible measurement. Using Cry3A-like toxin and CPTI, two genes with completely different insect resistance mechanisms, working together would greatly reduce the chances of an insect developing resitantance. Theoretically, the probability of an insect developing resitantance to two gene is the multiply of the probability of two independant genes.(Lin et al, 2008)

Biological Safety Device (BSD)

Inspired by our interaction with Tsinghua University at the Conference of China iGEMer Community (CCiC), we successfully designed our biosafety device, based on constructing a nutrient-deficient strain (see Human Practice for details).The modified bacteria will have the following characteristics:

• They can only survive in baits with exogenous added Diaminopimelic acid (DAP) or in the anaerobic environment of the S. Invicta gut.
• They will perish due to nutritional deficiencies when released into an aerobic environment without DAP.

• Our completed Project Safety Form can be accessed here.
• In order to conform to 2023 iGEM safety policies and rules, we have successfully submitted the parts that are not on the White List through the Check-in Forms before usage, including the following three types: toxic protein Cry3A-like , trypsin inhibitors CpTI (cowpea) and SKTI (soybean kunitz type).
• Additionally, to enhance laboratory biosafety and adhere to biosafety regulations, we opted to refrain from conducting animal experiments involving S. invicta. Instead, we validated the efficacy of our medication-engineered bacteria using literature evidence and mathematical models.