In early 2023, team members discovered that 90% of the willow trees near their school at Shahu Lake had been infested by an unknown pest. After consulting with the pest control expert, Professor Zhang Jiang, it was pointed out that most of these willow trees had been attacked by Plagiodera versicolora. This is a common forestry pest that primarily feeds on the leaves of poplar and willow trees, posing a certain threat to the forestry security in China. Professor Zhang also introduced us to some common methods of pest control: physical pest control, chemical pest control, and biological pest control. The team members developed a strong interest in this and conducted extensive literature review and group discussions.
We have learned that chemical pesticides are convenient, cost-effective, highly efficient, which provide quick results in controlling pests and play a crucial role in reducing pest damage as well as increasing crop yields1. However, the extensive use of chemical pesticides has led to serious environmental pollution. Chemical pesticides pose severe hazards to many non-target organisms, such as inhibiting the growth and development of amphibians2. Additionally, when chemical pesticides enter the water environment through leaching or other means, they can cause significant harm to aquatic organisms, resulting in irreparable damage to the entire ecosystem3. In recent years, the issues of environmental pollution and residual effects caused by the excessive use of chemical pesticides have become increasingly prominent. As a result, more and more chemical pesticides are being banned.Physical controls can be classified as passive (e.g., trenches, fences, organic mulch, particle films, inert dusts, and oils), active (e.g., mechanical, polishing, pneumatic, impact, and thermal), and miscellaneous (e.g., cold storage, heated air, flaming, hot-water immersion)4. It is environmentally friendly but has limited insecticidal range, low insecticidal efficiency and unstable insecticidal effect, which is unable to deal with large-scale or sudden insect pests. Biological insecticidal methods, such as transgenic plants containing BT protein genes, are environmentally friendly, which have good insecticidal effect and high economic benefits. But insects will develop resistance over time, and some studies have shown that BT protein may cause harm to human cells5,6.
After further exploration and consultation with our teachers，We decided to combine Plagiodera versicolora that is resistant to insects with RNAi technology, to cooperate with the construction of biocontrol bacteria that can effectively resist insects, so as to achieve better anti-insect effects.Keeping on the Do Not Release Policy, The iGEM team should not release any GMO or its products outside the laboratory. To ensure that our genetically modified microorganisms do not escape into the environment，in the highly unlikely circumstances of release, we designed a start switch for GMOs called lac operator(Control the mass synthesis of target dsRNA) and a photosensitive switch called KillerRed(It releases toxins under light conditions, causing the bacteria to lyse).The aim is to prevent its diffusion into the environment，and to better implement iGEM's safety and security policy and conduct experimental work safely and reliably.
Safety has always been the top priority for HUBU-WUHAN-CHINA. This year, we have followed the safety requirements of iGEM and implemented safety measures in line with other teams to prioritize the safety of ourselves and others in our project goals. With the assistance of teachers and staff from the School of Life Sciences at Hubei University, we have ensured that we can complete our project tasks to the best of our ability. In the laboratory, we have developed detailed experimental plans and strictly adhered to professional ethics to ensure that the experimental process does not harm our natural environment and the ecosystems that depend on it. We have submitted our experimental protocols to our PI and relevant teachers in the research group, discussed and confirmed that there are no biosafety issues with the experimental procedures in the laboratory.
2.1 Laws and regulations
All laboratories in our country must adhere to the requirements of the GB 19489-2008 standard and obtain certification from the Certification and Accreditation Administration of the People's Republic of China (CNCA). This year, our project was conducted in a laboratory with a Biosafety Level (BSL)-1. Throughout the entire experimental process, we fully complied with relevant laws and regulations, especially the general requirements for biosafety specified in the national standards of the People's Republic of China, as well as the Laboratory Safety Guidelines of Hubei University. We also fully adhere to the safety and security policies of the iGEM. We attach importance to laboratory fire safety, chemical safety, biosafety, radiation safety, large instruments and equipment safety, technical safety and accident emergency treatment. Before the experimental operation, all the team members received laboratory safety education in advance, and conducted the experimental operation in strict accordance with the equipment and experimental operation procedures.
2.2 Laboratory practice
2.2.1 Laboratory equipment, materials, drugs, and so on should be stored neatly,and the reagent bottles and related biological samples were labeled.It is forbidden to pile up or put it.The key of the laboratory should be kept by a special person, and the loan should be registered. No one can privately equip or lend it to others.
Figure 1. Neat reagent cabinet.
2.2.2 Before the experiment, the laboratory staff must carefully check all the instruments and equipment to confirm whether it is normal. After the end of the experiment, in addition to sorting out the instrument, it is necessary to turn off the power, close the faucet, the window, and lock the door.
Figure 2. The team members turn off the power supply.
Figure 3. Team dress code.
2.2.3 When performing experiments, you should wear a lab coat and wear appropriate personal protective gear.
2.2.4 The operation of volatile or toxic reagents should be carried out in the fume hood, and the experimenter should wear the relevant safety protective gear.
Figure 4. Fume hood.
2.2.5 After the end of the experiment, clean up in time and return the used items to their original places in order to keep the laboratory tidy and the smooth progress of the next experiment.
Figure 5. The team members are cleaning up.
Figure 6. Team members know the location of fire extinguishers.
2.2.6 Be familiar with the location of fire extinguishers, shower and eye wash devices and safety passages to ensure correct and safe handling in case of emergency.
2.2.7 Personal belongings should be kept in a special place in the laboratory.
Figure 7. Clothes are neatly placed.
Figure 8. Disinfection device.
2.2.8 The creation and use of genetically modified organisms will cause certain harm to the environment, so we will conduct timely elimination treatment of relevant experimental materials after the end of the experiment to prevent the flow of genetically modified microorganisms to the environment.
2.3 Bioassay Security
As stated by iGEM, the iGEM team should not release or deploy any Gmos or products of Gmos outside the laboratory. In order to greatly reduce the risk of release from our experiments, we have strict regulations on operating procedures and operating equipment. We have set up special operation areas, including insects feeding and testing rooms and ultra-clean workstations, and so on. When we enter the operation area from the outer chamber, we need to pass through a smart buffer chamber. The buffer room has two doors (entrance and exit). After the experimental personnel enter from the entrance and close the door, the buffer chamber will deliver clean air, which can prevent people from bringing pollutants in when they enter and keep the unclean air outside. After completing the above operations, the operator can open the exit door and come to the inner room (experimental operation area).
All experiments in this project were only carried out in the laboratory, and we should not take the strains away from the laboratory. After the completion of the experiment, we will promptly sterilize the strain and do a good job of hygiene and cleaning. Finally, we go to the outer chamber through the buffer chamber (the process is the same as when entering), which is more likely to prevent us from bringing out the pollutants in the inner chamber, and to ensure the safety and pollution-free of our experiment to a great extent.
Figure 9. Insect incubator.
Figure 10. Ultra-clean workbench.
Figure 11. Smart buffer room.
Microbiology classification
All of the microorganisms used in our project have been classified as risk Group 1 and treated in a BSL-1 level laboratory, which includes E. coli , P. chlororaphis B3-3G. We review the table of application levels for microbiological and biosafety laboratories pulished by American Biosafety Association (ABSA),as well as the Australian Group's list of human and animal pathogens and toxins used for export controls .They belong to the white list established by IGEM for safety, and these strains are already present in our laboratory inventory. Throughout the experiment, the strain was not taken out of the lab, so there was no risk of release into the environment. In addition, when dealing with strains, we use a separate ultra-clean workbench to eliminate any possibility of cross-contamination with other microbial species.
Summary and analysis of potential safety hazards of commonly used reagents and procedures in experiments
| Name | Potential hazard | Preventive measure |
|---|---|---|
| Alcohol | Highly flammable | Store them in a well-ventilated place |
| Chloroform | Corrosive properties, toxicity, and carcinogenicity | Wear protective gear and conduct local ventilation operation |
| Ethidium bromide | Mutagenic activity | Wear a protective gear |
| Hydrochloric acid | Corrosive characteristics | Operating in the fume hood |
| Caustic soda | Irritant and corrosive properties | Wear a good protective gear |
| Antibiotics (ampicillin, spectaculin, and kanamycin) | It is irritating when inhaled and exposed to high concentrations | Wear the personal protective equipment and learn the safety procedures |
| Ultraviolet rays | Mutagenic activity | Wear the necessary personal protective equipment |
Table 1. Common lab reagents and procedures and their hazards and protective measures
We take very seriously the potential impact that the engineered bacteria we build can have in the environment. Therefore, we mainly designed two safety control switches to ensure biosafety.
1. Switch one
We added a control switch to the plasmid expressing dsRNA——lac operator. The repressor protein produced by LacI binds to the related sequence, making it unable to transcribe and express foreign genes7.When added inducible primers IPTG (Isopropyl-beta-D-thiogalactopyranoside) for lac operator to the mediumlac, the repressor protein is allosteric and cannot bind to the related sequence,then the repressor is decommissioned8,11.This allows the cells to transcribe and express β-galactosidase (which hydrolyzes lactose to galactose and glucose and catalyzes the conversion of lactose to allolactose), permease (which tranquilizes lactose into the cell), acetyltransferase (which acetylates galactoside to form acetyl galactose), and target dsRNA9,10.At this time, foreign genes are heavily transcribed and efficiently expressed. Experexperiments, we controlled massive expression of target dsRNA by lac operator.
Figure 12. Schematic diagram of lac operator (after addition of IPTG, the repressor protein is allosteric and cannot bind to related sequences)
A)Lac operator vector map
Figure 13.KC3(actin+lac operator)
Figure 14.KC4(GFP+lac operator)
B) Experimental design
We named P. chlororaphis B3-3G that knocked out ribozyme III P. chlororaphis KC-P. The plasmid transferred to actin sequence was named KC1. The plasmid transferred to GFP sequence was named KC2. The plasmid transferred to lac operator on the basis of KC1 was named KC3, and the plasmid transferred to lac operator on the basis of KC2 was named KC4.
We performed Northern Blot to detect our lac operator switch. First, we cultured two bottles of P. chlororaphis KC-P to KC3 and P. chlororaphis KC-P to KC4 respectively,One of the two bottles of P. chlororaphis KC-P to KC3 and P. chlororaphis KC-P to KC4 was induced with IPTG (see Table 2). Then we extracted the total RNA of the bacteria and made a probe of the actin sequence, and finally performed Northern Blot.
| Number | Treatment condition |
|---|---|
| 1 | KC3 was transferred to P. chlororaphis KC-P and cultured，and added IPTG for induction，then total bacterial RNA was extracted for Northern Blot |
| 2 | KC4 was transferred to P. chlororaphis KC-P and cultured，and added IPTG for induction，then total bacterial RNA was extracted for Northern Blot |
| 3 | KC3 was transferred to P. chlororaphis KC-P and cultured,then total bacterial RNA was extracted for Northern Blot |
| 4 | KC4 was transferred to P. chlororaphis KC-P and cultured,then total bacterial RNA was extracted for Northern Blot |
Table 2.Northern Blot Test Experimental design
B) Experimental result
Figure 15.Northern Blot Test results (numbered 1,2,3,4 from left to right)
As can be seen from the experimental results, the lac operator switch can significantly inhibit the expression of target dsRNA. However, uninduced KC3 still has a small amount of color development, which may be due to the following two reasons: 1.lac operator is not a completely rigorous switch, so a small amount of dsRNA may still be expressed in the bacterial body; 2. Since we only added lac operator on one side of the RNA double strand, the single strand on the other side could still transcribed the target single strand RNA without IPTG induction. Although we added RNase to degrade single strand RNA in Northern Blot, a small amount of single strand RNA may still bind to the probe. So we decided to add a light-sensitive switch to the plasmid. After consulting with our instructor, we decided to add KillerRed light switch to further ensure the safety of our experiment.
2. Switch two
We designed the KillerRed switch to express the killerRed protein in the constructed engineered bacteria. KillerRed, a dimeric red fluorescent protein, was derived from a random and site-directed mutations of a jellyfish protein, anm2CP12,15,16 .KillerRed has a unique structure with a water channel to the chromophore that is responsible for its phototoxicity15,19.When red fluorescent protein is irradiated by visible white light, its structure will change and produce highly toxic ROS14,17,18. When ROS continues to increase, it can overcome the internal repair mechanism of cells, destroy cell components (lipid structure, proteins and DNA, etc.), and eventually lead to cell death13,17. The setting of the switch provides certain guarantee for the subsequent application. Because KillerRed is photoinduced to release toxins to break down cells, when ingested by bugs, the bacteria can still survive and maintain their insecticidal abilities, but with a high probability of preventing their disorderly spread in the natural environment.
We did experiments to verify our switches.
A) KillerRed vector map
Figure16.kc5(actin+lac operator+KillerRed)
Figure17.kc6(GFP+lac operator+KillerRed)
B)Experimental design
We designated the plasmid transferred into the KillerRed sequence based on KC 3 as KC 5. The plasmid transferred into the KillerRed sequence based on the KC 4 was named KC 6. Then we conducted the lethal efficiency verification experiment of KillerRed (see Table 3).
| Number | Group | Treatment Conditions |
|---|---|---|
| 3-3 | Bacterial control group | Did not turn to the killerRed switch and light for 3 hours |
| 2-3 | Bacterial experimental group | Turn to killerRed switch and dark culture for 3 hours |
| 1-0 | Bacterial experimental group | Turn to killerRed switch and light culture for 0 hours |
| 1-1 | Bacterial experimental group | Turn to killerRed switch and light culture for 0.5 hours |
| 1-2 | Bacterial experimental group | Turn to killerRed switch and light culture for 1.5 hours |
| 1-3 | Bacterial experimental group | Turn to killerRed switch and light culture for 3 hours |
Table 3. Experimental design for the validation of the KillerRed lethality efficiency
C) Experimental result
Figure 18.1 Bacterial control group 3-3
Figure 18.2 Bacteria Experimental Group 2-3
Figure 18.3 The bacterial experiment group 1-0
Figure 18.4 Bacteria Experimental Group 1-1
Figure 18.5 Bacterial Experimental group 1-2
Figure 18.6 Bacterial Experimental group 1-3
Figure18.Experimental results for verifying the lethal efficiency of the KillerRed
According to the results of the experimental group 1-2, it can be seen that more than 95% of bacteria can be killed by light for more than 1.5 hours, and the effect is significant. There are still very few colonies alive, but as iGEM says, there is no absolute safe switch. At this point, the construction of our plasmid dual safety switch was successfully completed. Lac operator switch can control a large amount of dsRNA expression. KillerRed switch can kill microorganisms with this gene under the condition of light, so that biocontrol bacteria will not flow into the natural environment,which could ensure the safety of production and use process.
In addition, we added resistance genes to the constructed dsRNA expression plasmid to ensure that the plasmid was not lost. When engineered bacteria are released into the environment, plasmids are gradually lost due to the absence of resistance selection pressure20,21. It reverts to a wild-type state, reducing its negative impact on the environment even more.
For now, this is just a laboratory experiment designed to scientifically advance the prospects of genetically modified organisms and synthetic biology in the insect-resistant market. All in all, on the whole, Plagiodera versicolora and the dsRNA or other substances expressed by us are environmentally friendly and will not cause harm to the natural environment.
Expectation
We have determined the preliminary anti-pest scheme and verified its high efficiency against the original biocontrol bacteria in the experiment. RNAi technology is highly targeted to target pests, and has no harm to crops, humans and livestock. Moreover, many studies have pointed out that Coleoptera insects are extremely sensitive to RNAi technology, and Coleoptera, as the largest insect order, is known to have 350,000 species, and many of its phytophagous species are agricultural and forestry pests, posing a great threat to agriculture and forestry. So in theory, RNAi technology might be a Nemesis against Coleoptera pests such as Plagiodera versicolora. In addition, the two safety switches we set up can also effectively control the current possible unknown effects of dsRNA and biocontrol bacteria on the environment, preventing problems before they occur. All kinds of data can show that our existing work has contributed to promoting the green and environmental protection of pesticides and maintaining the sustainable development of the natural environment.
This is despite the fact that our insecticidal programs are currently less effective and easy to use than chemical pesticides. However, in the long run, when resistance emerges, the replacement of new available chemical pesticides consumes high research and development costs and a long safety assessment cycle. In addition, chemical pesticides in the production process also involves the mining of salt mines, and will cause a lot of energy consumption and a certain amount of emissions. The above seems to be the difficult situation that chemical pesticides are difficult to avoid in the development and production. But a synergistic solution between RNAi and insect-resistant P. chlororaphis B3-3G seems to be the answer. Thanks to the fast and convenient sequencing technology and the flexibility of RNAi technology, in the face of sudden resistance problems or sudden pests, if you want to change the target sequence or target organism, you only need to sequence the target. After the initial production line is completed, the cost only includes gene sequencing, whether it is independent sequencing or sequencing in relevant institutions is very convenient. There is no need to replace the production line equipment and re-evaluate the safety of the equipment. Therefore, under the premise that both biopesticides and chemical pesticides have high anti-insect effectiveness on pests, the synergistic scheme of RNAi and insect-resistant Plagiodera versicolora has higher benefits in the development.
Our expected result is to construct a biocontrol bacterium that is highly effective against Plagioderaversicolora(the dsRNA-expressing P. chlororaphis B3-3G ).The use of RNAi technology for entomology research and pest control has become a research hotspot. This technology is highly targeted, harmless to crops, humans and animals, and has good application potential. Due to its advantages of low environmental pollution, strong killing specificity and high selection flexibility, biological control has a good prospect of research and development and market. The biocontrol bacteria constructed by us is relying on biological control means to achieve the purpose of protecting poplar and willow, reducing the use of chemical pesticides and effectively resisting insects. With the rapid development of science and technology, the anti-pest efficiency of the RNAi and P. chlororaphis B3-3G collaborative anti-pest program will continue to improve and optimize.Finally achieve our sustainable development goals of both plant protection and ecological civilization!
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