Team:CAU China/Implementation
Proposed Implementation Overview
Considering the increasing saline-alkaline soil problem globally, CAU_China 2021 team plans to construct a kind of engineered bacterium using Corynebacterium glutamicum as the chassis and put it into the soil to work.
The proposed product of our project is a kind of bacterial agent, which means our engineered bacteria will be stored in freeze-dried powder, solid (particle, bacterial powder), bacterial liquid or other mediums to apply to different storage and service environment. Facing different types of saline-alkaline soil, such as heavy alkalinity, heavy salinity and soda saline-alkaline soil, we plan to design different bacterial agent proportioning schemes and use them in the form of different packages according to local conditions.
Besides, to allow users to know their soil condition before using our microbial agent, we also plan to develop a kind of test strip that can quickly measure the salt concentration and pH of soil, on the basis of the gsiB promoter and atp2 promoter we have verified in our experiments.
End users
Our target users involve workers in soil restoration industry, forestry, agriculture, individual farmers, and even anyone who has the need for planting.
For individual farmers and workers in agricultural industry, saline-alkaline soil puts high stress to plant roots, which is not good for plant growth or agricultural economic increase. Our bacterial agent product is designed to reduce salt and alkali stress, to optimize soil mass structure and therefore create better environment for plant growth, increasing the grain yield. Hence, individual farmers and employees in the planting industry will be the direct end users of our project.
For workers in soil restoration industry or environmental protection, the current soil restoration methods are labor-consuming and expensive. We expect that the bacterial agent product can directly repair saline-alkaline soil, and effectively prevent secondary salinization by using the excellent properties of γ-PGA, which will greatly improve the efficiency and convenience for soil treatment, reduce the price of soil restoration, and allowing broader access to better soil in poor regions. This is a great blessing for workers in related industries.
For the ordinary people who need to plant something, they can also sprinkle our bacterial agent around the plant roots. Due to the role of additional substances in the bacterial agent, the bacteria can secrete sufficient polyglutamic acid before the salinity and alkalinity drop to the threshold, interacting with plant root system to promote plant growth. Therefore, those people with planting needs are also one of our end users.
Usage
We envision others using our soil test strips first to determine the salinization and alkalinization level of the soil sample (they can surely skip this if they get high-tech device). Then, we envision them using the particular form of our microbial agent according to local environment and the soil state, and they should also give reasonable irrigation at the same time.
There are four forms of our bacterial agent, including freeze-dried powder, powder, granular agent and liquid agent, and we envision users applying them in different conditions.
If users can afford the high price, they can choose freeze-dried powder bacterial agent, which is easy to store and can easily seep into soil. When using, they need to mix the agent with special liquid in advance.
If the using condition is greenhouse or other places which is not windy and dusty, users can use the powder bacterial agent by application through holes or trenching. The biggest advantage of powder is that it has a long storage time and is easy to transport.
If users need the agent to both be long-time storage and suitable for windy and dusty area, they can use the granular agent by trenching instead.
If users want to achieve the best effect with low quantity of our product, they can try the liquid bacterial agent by applying it through sprinkler irrigation or drip irrigation. Particularly, they need to use our product as soon as they receive it because the liquid agent can’t be storage for a long time.
Safety
Our bacterial agents are applied in the natural environment, so we need to consider the safety problems such as gene drift and whether it will pose threat to the growth of normal bacteria in the soil. As a result, we set a kill switch to ensure that the engineering bacteria immediately die after the successful treatment of saline-alkaline soil.
We used the salt-induced promoter Patp2 and the alkali-induced promoter PgsiB to construct an OR gate. The downstream of the two promoters were connected to the repressor protein, and the downstream of the promoter acted by the repressor protein was connected to the toxin gene ndoA. When salinity or pH was high, both salt promoter and alkali promoter will express repressor and inhibit the transcription of ndoA. Only when the salinity and pH decrease to an expected level, the expression of ndoA will go up and its product can disrupt the gene expression by cutting mRNA, resulting in the death of the bacteria. To learn more, please see design: kill switch for details.
Meanwhile, we also need to consider possible dual-use. Though we think our aimed product, γ-PGA, and the bacterial agent product will be harmless to the environment, we still can’t ignore the possibilities of malicious modification of its gene. Means to prevent dual-use need to be further developed in our project afterwards.
Challenges
Our product contains genetically modified bacteria. Although they have no resistance to antibiotics and have a kill switch for protection, they may still have a certain impact on the natural environment. If our bacterial agents are produced and utilized anywhere outside the laboratory, extensive tests need to be carried out to ensure the effectiveness of the suicide switch and absolute safety to the environment and human beings.
In addition, the specific formula of bacterial agent has not been determined yet and needs to be optimized. We also need to communicate and negotiate with relevant enterprises to ensure that our products will have an absolute advantage in price and accessibility.
Moreover, China has passed a new law on biosafety, which has entered into force on April 15, 2021. This law clarifies the risks of biotechnology, laboratory safety and microbial drug resistance. It will also have provisions to prevent and respond to specific biosafety risks, including the research, development and application of bio-products. Therefore, we need to consider whether our bacterial agent is in line with the law and can be merchandised. Plus, we need to apply for governmental approval in order to keep our activities legal before actually developing our bio-products.
Finally, because our end users include non-professionals such as individual farmers and ordinary people with planting needs, we must give extremely clear guidance on how and where to use our product, and do a good job in relevant risk assessment to prevent any biosafety problems caused by vague instruction.
If users need the agent to both be long-time storage and suitable for windy and dusty area, they can use the granular agent by trenching instead.
If users want to achieve the best effect with low quantity of our product, they can try the liquid bacterial agent by applying it through sprinkler irrigation or drip irrigation.
Particularly, they need to use our product as soon as they receive it because the liquid agent can’t be storage for a long time.