Engagement in safety
Safety and security trainingWe received safety and security training from our advisor and iGEM ambassador Wenxi Li when we first set up our team. |
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Participating in a safety workshop hosted by iGEMWe participated in CCiC iGEM Responsibility (including Safety and Security Program and Human Practices Program) event, learing many safety rules in iGEM competition in Dual-Use Research Workshop of the meeting. |
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We interview many stakeholders in our province to learn the use of biological drugs in paddy fields, for instance, an professor in Shenzhen Comprehensive Experimental Base of Chinese Academy of Agricultural Sciences told us our engineered fungi may cause harm to small animals in fields so that the control of dosage is necessary. |
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In addition, we sought help from Agricultural Science Institute of Guangdong Province and local areas to evaluate hazards or risks of our project. |
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We will also keep in touch with officers in governments in order to learn policies, laws and regulations of biopesticides and field testing devices. |
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Managing risks and hazardsDuring the project, we communicated with our PIs in time, who specialize in plant conservation and genetic engineering. And we will keep consulting with stakeholders about managing risks in future, paying attention to safety throughout the process. |
Consulting Biopesticide Professor
In order to ensure the safety of the future application of Trichoderma engineering bacteria, we
have an email interview with Dr.Luo——a professor of biopesticide. |
Lab safety
During the whole process of the project, all our experiments were strictly carried out in the laboratory. We conducted our experiments in specific zones and equipment, including biosafety cabinet, specialist greenhouse, chemical fume hood and so on. We wear personal protective equipment (lab coats, gloves, eye protection, etc.) in lab. Considering biosafety, we only conducted experiments on these organisms in the laboratory. First, we wear protective measures to keep ourselves safe. In addition, we carried out sterilization in time to prevent the strain from escaping outside the laboratory. What is more, we collected the leave and stems of rice crops, using them to simulate the onset and control of rice sheath blight inside the lab instead of in the field. Trichoderma spps produce spores, which cause risks to humans if inhaled. Rhizoctonia solani do not produce spores. However, we are supposed to take measurements in order to prevent them from leaking into environments, causing harm to plants. Especially for the organisms in our check-in form, all of the experiments involving them were conducted in our biosafety cabinet.
We strictly followed the laboratory safety rules of Shenzhen University and the safety guidance provided by iGEM. On this basis, all team members attach great importance to laboratory safety.
Project safety
Experimental organisms
We have already conducted experiments and verified that Trichoderma atroviride is able to control the pathogen Rhizoctonia solani effectively. In addition, we have read articles about Trichoderma atroviride, which reported that this fungus species is able to colonize the rhizosphere of crops to help them survive and resist diseases. Rhizoctonia solani is the pathogen of rice sheath blight, but it is safe because of its low risk of harm to humans and the environment. We also selected common laboratory strains of Escherichia coli, with rich experimental experience of Escherichia coli.
To ensure that the risk groups corresponding to the organisms we work with was appropriate for our experimental environment. We first consulted the List of pathogenic microorganisms transmitted from humans, China CDC. Trichoderma atroviride，Trichoderma reesei，Rhizoctonia solani AG-1 and Rhizoctonia solani AG-3 were not found among the fungal agent as BL2-4 by China CDC. Therefore, we believe that the above four fungi belong to BL 1 class organisms.
To further ensure that the organisms we work with were safe and reliable, we reviewed the NIH-Guideline. Similarly, none of the four organisms we work with were found in the RG2-RG4 list of Fungal Agents in NIH-Guideline, further confirming that they are safe in common experimental Settings.
The mission of our engineered organisms
Our fungi (Trichoderma atroviride) will be engineered to block the transmission of pathogens in aquatic interface of rice field. They will overexpress hydrophobic proteins epl1 and proteases prb1 as well as express plant-derived antimicrobial peptides sn1, which makes them more competitive to the pathogen Rhizoctonia solani. Their mission is to block the transmission of pathogens, so we will test them on isolated leaves and stems of rice (wild type) inside our lab.
Our bacteria (E.coli Ht115(DE3)) are engineered to produce and release shRNA molecules which are able to interfere the essential genes in R.solani infection. They (E.coli Ht115(DE3)) are meant to produce and release RNAi molecules. We have tested these RNAi molecules in wild type rices inside our lab.
Parts
Engineered Trichoderma atroviride for prevention: our engineered Trichoderma atroviride express epl1 and prb1 which are derived from Trichoderma spp. In addition, sn1 is secreted to fight pathogens and derived from potatoes. Thus, these genes are safe for rice and field ecosystems.
RNAi molecules for treatment: For biosafety, the candidate RNAi fragments were submitted to the total mRNA database for blast, and the sequence similarity was compared. Focus on species with more than 90% similarity and their nucleic acid fragments to ensure that there is no matching of common species (human, rice, dog, wheat, etc.) to ensure the specificity of the sequence.
Suicide switch
Considering safety, T. atroviride is a Class 1 organism according to the ATCC (American type culture collection) classification, which means it is safe under almost all conditions. However, in order to prevent our engineered fungi from escaping into the environment and causing adverse effects.
1. Firstly, we designed a condition-triggered suicide switch for T.atroviride.
- In the lab and inside the wrapping materials, we cultured T. atroviride under glucose-rich conditions. Glucose within high concentration inhibited the cbh1 operon, rendering this suicide pathway unexpressed.
- When T. atroviride are released to the field and glucose concentration gradually reduces in the the wrapping materials and surrounding environment, the inhibitory effect of glucose on the suicide switch reduces correspondingly. At the same time, the utilization of carbon sources by T. atroviride converts to many kinds of saccharides such as cellulose and sophorose. These saccharides induced the expression of cbh1 operon, leading to the suicide of T. atroviride.
2. Besides, we came up with a tentative idea of timed suicide switch including oscillator and effector.
- The oscillator is a periodic expression device based on the principle of reciprocal inhibition of three repressor proteins. The effector is a suicide device based on MazEF system. The two systems are linked by a repressor protein. In our design, antitoxin MazE is maintained at a stable concentration in cell while the concentration of toxin MazF increases with periodic fluctuation corresponding to the oscillator. When a certain period is reached, the concentration of MazF will be above MazE, initiating the programmed cell death of T. atroviride.
This suicide switch is different from the one above, which is our preliminary design for future applications of engineered Trichoderma. Please see the "Safety in future application" section for more details of this suicide switch.
Safety in future application
If our project can continue in the future, we will carry out experiments in certain experimental fields under safe conditions to test the prevention or detection or treatment performance of each product (E-nose, LAMP-LFD, sustained-release tablets containing engineered Trichoderma powder, RNAi spray). We will also communicate closely with stakeholders and actively cooperate with them to understand the approval processes and regulations related to biopesticides and field testing devices, such as probiotics related regulations, biological drug production and review process, etc.
In addition, pesticides are an indispensable tool for farmers and are used as an effective and beneficial tool for pest management in most agricultural production sectors. However, when mixing and applying products or working in treated fields, there are always hazards and associated risks associated with exposure by farmers and professional applicators, mainly through skin exposure, absorption and inhalation. So in the future implementation, we recommend that our users must take routine precautions when spraying to prevent inhaling our product. For example, use appropriate personal protective equipment (PPE) for protection.
A tentitive timed suicide switch
How to ensure the safety of engineered microorganisms for agricultural applications is a serious issue. In order to prevent the engineered microorganisms from escaping, it is a feasible method to use the conditional triggered suicide switch. However, this is not foolproof, because the triggering conditions in the natural environment are uncertain, and there may still be the possibility of escaping. 2022 SZU-China attaches great importance to the safety of engineered microorganisms and hopes to design a suicide switch activated by endogenous factors.
Based on the classical gene oscillator, we propose a concept of timed suicide switch, hoping to kill the engineered microorganism population at a certain time. Based on the classical model organism Escherichia coli, we have designed and improved the gene circuit. We also explore the properties of the timed suicide switch through a mathematical model.
Figure 1. The tentative timed suicide switch based on classical gene oscillator.
Oscillator
Gene oscillation is a gene regulation mechanism, and the amplitude and period of oscillation reflect the gene expression. Its principle is that three gene modules whose encoded repressors inhibit each other are connected in series to form negative feedback, and the periodic change of the content of repressor is realized by the inhibition and deinhibition of gene modules. The oscillator contains three repressor proteins: TetR from the Tn10 transposon, λ cI from bacteriophage λ, and LacI from the lactose operon. Each repressor carries an LVA degradation tag at the C-terminus. The oscillator device is encoded on a low copy plasmid pSB3C5.
Effector
The core of the effector is the MazEF toxin-antitoxin system from Bacillus subtilis, which can lead to programmed cell death. The antitoxin MazF is controlled by the Promoter tetR, and the toxin MazE is controlled by the constitutive Promoter PJ23110. MazF is an endonuclease that specifically cleaves UACAU sites on mRNA. MazE combines with MazF at a ratio of 1:1 to occupy its active site and make it lose its toxicity. The effector is encoded in the high-copy plasmid colE1.
Functioning
The engineered bacteria with a timed suicide switch were placed in an IPTG-rich medium or in a dormant state before being applied in fields. The purpose of being placed in IPTG is to continuously activate the PlacI and make the oscillator unbalanced and stagnant, in which circumstance MazF does not express.
After being applied to the field, the oscillator is re-activated with the release of IPTG and the resuscitation of the engineering bacteria. The contents of three repressor proteins changed cyclically: lacI inhibited the expression of tetR, tetR inhibited the expression of λ cI, and λ cI inhibited lacI expression. That is, the three promoters PlacI, PtetR, and PλcI were alternately activated.
Figure 2. Mechanism of the tentative timed suicide switch
As for the effector, MazE was constitutively expressed and maintained at a certain concentration in the cytoplasm, while the expression of MazF was inhibited by tetR and showed a fluctuating increase. In a simplified model, MazE and MazF bind at the ratio of 1:1, resulting in toxin inactivation. When the concentration of toxin MazF is higher than that of antitoxin MazE, the extra toxin MazF plays the role of endonuclease to cut mRNA and kill the engineered microorganisms.
1. The number of effector plasmids fluctuates at a high level
First, in the timed suicide switch1.0, the oscillator is placed on a rigor type plasmid and the effector is placed on a relaxation type plasmid. The two devices are in a separated state and communicate with each other through tetR (tet repressor). Due to the poor replication control of effector plasmids used, the number of effector plasmids fluctuated at a high level, and the number of effector plasmids in each cell of the engineered bacteria population was not strictly the same. This difference sets the activation and inhibition of Promoter tetR on the oscillator and effector at variance, making it difficult to achieve synchronous oscillation of toxin mazF's concentration in the population.
Therefore, we transferred the MazF expression device from the effector plasmid to the low-copy oscillator plasmid, which could greatly reduce the amplitude standard deviation of MazF oscillation and help to achieve the synchronization of engineering bacteria.
2. Overloading of Proteolytic System in engineering bacteria
Secondly, for the purpose of realizing the rapid oscillation, the C-terminal of the three repressor proteins of the oscillator in the timed suicide switch1.0 is added with a rapid degradation tag. However, because these repressor proteins with the tag share the same Proteolytic System with other proteins, this leads to an overload. Theory shows that the supersaturation of the Proteolytic System will lead to large random fluctuations in the oscillation of individual cell, and the fluctuation caused by the delayed degradation of repressor will be fed back to the repressor protein through the Proteolytic System, thus interfering with the oscillation.
Therefore, we deleted the LVA degradation tag at the C-terminus of repressor proteins, eliminating the degradation competition between repressor proteins, which will reduce the noise of single cell oscillation and prolong the oscillation period. This will also extend the suicide time of engineered bacteria, so that the engineered bacteria can survive longer, which is good for our project.
3. Low disinhibition prefabricated of Promoter tetR
Third, previous studies have shown that the noisiest phase of oscillations is when the level of TetR is the lowest. At this time, the disinhibition of Promoter tetR occurs at a very low threshold of tetR. Theory suggests that if this threshold is raised, the regularity of oscillations may be greatly improved.
We added five highly efficient tetR binding sites to the effector plasmid. The modified effector plasmid can be used as a sponge of tetR to adsorb tetR. The tetR binding site was derived from Promoter tetR and artificially engineered to increase binding efficiency. In theory, the introduction of tetR molecular sponge will reduce the effective concentration of tetR in the cytoplasm, thus increasing the apparent threshold of Promoter tetR disinhibition and greatly improving the regularity of the oscillator.
Figure 3. The tentative timed suicide switch2.0
To sum up, the upwards improvements have been made to the tentative timed suicide switch, creating the stable and elegant timed suicide switch2.0.
Predicting
In theory, the oscillating rise of MazF concentration and the stable concentration of MazE can be predicted by the mathematical model. That is, we can predict the exact time point of suicide as long as we have accurate parameters. We performed a modeling analysis of the timed suicide switch1.0, mathematically demonstrating that the engineered bacteria will begin to suicide after approximately 770.1 minutes, when the concentration of toxin MazF is higher than that of antitoxin MazE reversely.
Figure 4. Predicted results of the timed suicide switch1.0.
See more about models of the timed suicide switch in model.
Furthermore, we may also be able to achieve different suicide time by changing the parts of the gene device. For example, by replacing the constitutive promoter with different transcriptional activity, we can change the stable concentration of antitoxin MazE, so as to change the time of concentration reversal of MazF and MazE. Thereby changing the suicide time of the engineering microorganisms.