Our project this year aims to address the global ecological security and economic issues arising from the widespread use of herbicides. We offer an environmentally friendly biofertilizer that attempts to solve these problems through synthetic biology. An engineered Escherichia coli is being constructed to produce a novel herbicide, aspartic acid and extracellular polysaccharide (EPS), under blue light and can be released into the soil in a controlled manner at a high temperature of 42 ° C, avoiding overuse of herbicides and possible residue, while promoting water retention and sand fixation of EPS. The intelligent synthesis and release of our biofertilizers will maximize the effects of herbicides and EPS, contributing to the environment and society.

Project production design

According to our design, the final product is an engineered bacterial powder containing a precursor synthesis plasmid and a multifunctional plasmid. The precursor synthesis plasmid converts glucose into the key precursor GPP, while the multifunctional synthesis plasmid is responsible for blue light induction and regulation, product AA and EPS synthesis, and temperature control of bacterial suicide lysis. After a survey of the use of herbicides in the world today, we chose to synthesize new herbicides and designed an additional circuit to synthesize exopolysaccharide EPS. The use of healthy and environmentally friendly exopolysaccharide cross-linking to form a "biofilm", which plays a role in soil water retention and soil consolidation, reduces the amount of plastic film used. Initially, E. coli was grown in an LB medium resistant to carna and chloramphenicol. The engineered bacteria were diluted after growth to the logarithmic stage, and the concentration of the bacteria was determined by blood count and colorimetry, respectively. E. coli was diluted to 5-10 MCG/ml. The engineered fungus powder was evenly sprayed on the surface containing the weed at a concentration of 200 mg/m 2. Depending on soil conditions, sodium alginate can be added to bacterial solutions to improve the compressive strength of biological crusts and increase the growth efficiency of the initial symbiotic system. When the fungus powder enters the soil and is irradiated with blue light for a certain time (about 4h), the concentration of bacteria and downstream products will be significantly expressed, and the yield will be increased (specific data can be seen in the modeling results). The temperature-controlled thallus lysis system will be expressed when the soil temperature is higher than 42°C. According to the data, such a temperature distribution is just enough for engineered bacteria to grow and produce products at night, and the thallus will gradually lysate and release products to act on weeds and soil at around 10:00 am. Such results are in line with our expectations for the intelligent and sustained release of products.

The audience for our products is herbicide manufacturers on the one hand and individual farmers on the other. The advantage of cooperating with manufacturers is that once the safety production standards are met, our products can be put into production in large quantities for application, which will play a very important role in the optimization of herbicides and global environmental protection. For the individual farmers, we are after the field investigation and feel their fear and discontent with the existing herbicide, and delighted to discover they are interested in our solution is, perhaps the product itself will not our direct sales, but we are willing to give away and try to provide farmers with our products.

Biological safety

In terms of biosafety, the E. coli we use are classified as biosafety Level 1 (BSL-1) and they are not harmful to humans. They are also strains that are already present in the natural environment and will avoid the risk of species invasion. At the same time, the plasmid vectors PGPP and PSB1K3 were respectively low-copy and medium-high copy to reduce the probability of gene leakage or mutation caused by excessive production pressure. In addition, we considered possible strain and gene leakage, but with our temperature-controlled kill switch, escaping and spreading E. coli will die, ensuring both lysis and release of product and safety.

Challenge

In the process of project design and experiment, we inevitably encountered some challenges. We also need to test the period and production areas of the strains inoculated in the actual soil environment, so that they can achieve maximum efficiency. Moreover, under natural conditions, our engineered bacteria run the risk of gradually losing their plasmids. Engineered bacteria without plasmids are unable to produce AA and EPS normally and efficiently, so they cannot perform this function as intended. However, we can avoid this problem by integrating the built circuits into the genome with genome editing tools such as CRISPR-CAS. The temperature varies greatly under natural conditions, and climate differences in different agricultural areas also need to be considered. Whether our temperature control switch can function properly is still a problem that needs to be solved.