Food security is fundamental to people's livelihood and is also a research hotspot in iGEM. This project is committed to developing a method for the efficient synthesis of the green pesticide 5-ALA using synthetic biology technology, and contributing to the alleviation of the world food crisis. ALAS is the key enzyme for 5-ALA synthesis, we screen for highly active ALAS genes and modify them. A CRISPR-related transposon system was used to integrate ALAS genes into the Escherichia. coli genome for stable expression. Further, we used enzyme-constrained models to predict and up-regulate new key targets related to 5-ALA synthesis, and found some new effective targets. In addition, we use droplet microfluidic high-throughput screening technology to help us quickly screen for high-yielding strains.

New Technology

1.CRISPR- associated transposon (CAST) system

The stable expression of ALAS is an important prerequisite for the efficient synthesis of 5-ALA. However, during the experiment, we found that the free expression plasmid carrying ALAS was easy to be lost, which indicated that the expression strategy of the free plasmid was unstable, and it was difficult to achieve stable and efficient expression of ALAS in the BL21 chassis strain of E. coli. Therefore, in this project, we used CAST system [2] to directly integrate the ALAS gene into the E. coli genome to improve its genetic stability [3]. The system combines the RNA-guided nature of CRISPR technology with the DNA mobility of transposons, enabling precise insertion of large DNA fragments at specific loci in the genome without generating DNA double-strand breaks. The team constructed the PQcasTns plasmid carrying the Tns transposase gene and array sequence [4] and the PtDonor plasmid carrying the ALAS gene, respectively, and used these two plasmids to integrate the ALAS gene into the E. coli genome in multiple rounds to achieve stable expression of the gene. The experimental results show that the precise genomic locus integration strategy of ALAS gene mediated by CRISPR transposable technology not only creates stable experimental conditions for our team's follow-up research, but also provides a new reference method and reference for other iGEM teams to stably express foreign genes.

Fig.1 Schematic diagram of the CASTs experiment

2. Enzyme-constrained model

Through literature review, we found that the current targets for increasing the yield of 5-ALA focus on the enzymes that directly mediate the synthesis of 5-ALA, while ignoring other key regulatory targetsThis limits the further increase in 5-ALA production. To overcome this bottleneck, we used enzyme-constrained models to identify several novel targets. Based on the enzyme-constrained ec_iECBD_1354 model we previously constructed in Escherichia coli BL21, we introduced reactions related to 5-ALA synthesis to successfully predict targets that need to be upregulated and downregulated. The enzyme-constrained model not only has good prediction accuracy, but also shows the potential to guide the modification of genetically engineered bacteria. By applying this model, we hope to provide ideas for future iGEM teams to predict the targets that need to be engineered.

Fig.2 Process diagram for constructing ec_iECBD_1354

New Method:

Microfluidic High-Throughput Screening Technique

1. Self-designed PDMS microfluidic chip

In view of how the transformed engineering bacteria are encapsulated into the droplets and carried out subsequent screening operations, we used AutoCAD to independently design a schematic diagram of the PDMS microfluidic chip, and commissioned the production to obtain the actual product. The PDMS chip is designed to make the production and sorting of microfluidic droplets. This chip is economical, fast, and efficient, and easy to observe. Future iGEM teams can use similar designs if they need to apply the microfluidic screening.

Fig.3 PDMS generating chip design drawings and physical products Fig.4 PDMS sorting chip design drawing and physical products

2. A complete procedure of droplet high-throughput screening

During the year, our team explored the right oil phase (fluorinated oil) and surfactant, as well as the optimal flow rate for the inner and outer phases, to generate droplets of uniform size, stability to support long-term growth and subsequent screening of strains. In addition, we use absorbance activated droplet sorting (AADS) technology to combine absorption spectroscopy with microfluidic technology to distinguish the biomass of droplets by measuring the absorption intensity of droplets to 600 nm light, so as to sort droplets with high E. coli concentrations, which has a wide range of universality. We hope that the exploration and ideas for high-throughput screening of droplets can help the iGEM team apply this technology in the future. When they encounter related difficulties, we believe it may give them some inspiration.

Fig.5 The complete procedure of droplet high-throughput screening

Food is essential for the survival of every human being in the world, yet today we can still see that many people in the world are hungry. As an excellent biological pesticide, 5-ALA can improve the stress resistance, promote growth and yield of food crops, and is biodegradable, green and environmentally friendly, and will not cause pests to develop resistance. Therefore, accelerating the popularization and application of 5-ALA in agriculture will help alleviate the food crisis, protect the environment, and promote sustainable development.

Our human practice starts with research. Visiting and issuing questionnaires helped us to summarize the direction and goal of practice. We made rich efforts in science education and synthetic biology popularization, and achieved good results. At the same time, we have established good partnerships with other iGEM teams, enterprises and scholars.

Fig.6 A general overview of the contributions of dry lab

We are eager to introduce synthetic biology and green biopesticides to more people through our projects, especially to cultivate the interest of children and teenagers to arouse their attention, and convey our concept of sustainable development, so as to contribute to creating a rich, harmonious and sustainable future.

1. Cui, Z., et al., Stable and Efficient Biosynthesis of 5-Aminolevulinic Acid Using Plasmid-Free Escherichia coli. Journal of Agricultural and Food Chemistry, 2019. 67(5): p. 1478-1483.

2. Vo, P.L.H., et al., CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering. Nature Biotechnology, 2021. 39(4): p. 480-489.

3. Yuan, Q. and X. Gao, Multiplex base- and prime-editing with drive-and-process CRISPR arrays. Nature Communications, 2022. 13(1): p. 2771.

4. Durrant, M.G., et al., Bridge RNAs direct programmable recombination of target and donor DNA. Nature, 2024. 630(8018): p. 984-993.