Introduction When we decided to use phage therapy to solve the problems caused by Xanthomonas in agriculture, we conducted a large number of investigations and found that the existing phage therapy had the following problems:

1. Phage therapy has a short action time, and bacteria will quickly enter into the growth phase in the actual environment.

2. Phage therapy is highly targeted and usually requires a mixture of multiple phages to treat diseases caused by multiple bacteria.

3. Bacteriophages sprayed directly cannot enter the inside of the plant and will quickly decompose outside.

4. The cost and preservation problems of synthetic phage.

5. When phages are released, the spread and growth of bacteria can only be estimated by appearance.

Therefore, in our project, we focused on several major problems of phage therapy, conducted research on the phage species of Xanthomonas, and proposed some effective solutions.
1. Dsf sensor design (BBa_K3820020) 1. Purpose:
DSF sensor system is used to specifically detect the information quorum sensingmolecules of Xanthomonas (Gram-negative bacteria) before Xanthomonas enters the logarithmic growth phase.

2. The quorum sensing system
The quorum sensing system, guided by DSF signaling molecules, is widespread among Xanthomonas species. Therefore, we conducted research on the quorum sensing system in the targeted bacterial strains and the conclusions are as follows:

The DSF molecule is a sensor molecule used by X. Fastidiosa to communicate between the same species. It is a 2-cis unsaturated fatty acid. The DSF induction system consists of two basic systems, including RpfC and RpfG. RpfC is a histidine protein kinase that can be activated by DSF molecules, which can then activate RpfG. RpfG is a phosphodiesterase that can convert the second messenger c-di-GMP into c-GMP. cAMP Receptor-Like Protein (CLP) is a transcription factor that regulates the production of mannanase (ManA). ManA is negatively regulated when c-di-GMP is combined with CLP. Therefore, the reduction of c-di-GMP caused by DSF molecules will lead to more ManA being transcribed.

Figure 1:Quorm sensing design
3. About Vc2 riboswitch
By searching in the iGEM original library (parts library), we successfully found the original part uploaded by the Wageningen_UR team in 2019 who used it to sense DSF molecules. The riboswitch, Vc2 riboswitch (BBa_K3286202) can sense and bind to the second messengers in the cell. We discovered how to utilize the Vc2 riboswitch as the switch of the DSF molecular counterpart.
Figure 2: c-di-GMP riboswitch from V. cholerae. a. Secondary structure of the riboswitch upstream of the tfoX-like gene in V. cholerae. The P1 helix is shown in dark blue, P2 in light blue, P3 in green. The asterisks next to C44 and G83 indicate that these residues are base-paired. Nucleotides that directly contact the bases of c-di-GMP are shown in yellow. c-di-GMP is shown in orange.

b. Crystal structure. The U1A protein used for cocrystallization has been removed for clarity. Coloring is the same as in part a. (Smith & Strobel, 2011)
4. About the plasmid design: Based on the design of Vc2 riboswitch, we plan to use synthetic biology methods to test the effectiveness of Vc2 riboswitch in Xanthomonas and Xanthomonas oryzae. The design pathway is as follows (BBa_K3820020). Figure 3: DSF sensor design Figure3: In the detection design of the DSF sensor system, we used a high expression promoter: J23108, and added a special RBS sequence in Xanthomonas oryzae: BBa_K3820010. In the downstream of Vc2 riboswitch, we added the EYFP (BBa_E0032) reporter gene to monitor the effectiveness of Vc2 riboswitch. Design consideration:

From the above regulation of the Xanthomonas DSF system, we can know that the pathway also contains Rpfc membrane protein and Rpfg protein. However, in this year's project, we decide not to edit the Rpfc protein and RpfG protein into the plasmid through synthetic biology and control their expression in Xanthomonas. This is because our engineering bacterias, Xanthomonas campestris and Xanthomonas oryzae, both have a certain amount of conventional expression to detect the external environment and their own unique Rpfc and RpfG proteins. Through previous research, we found that the working pathways of these proteins are different in different types of bacteria, so adding RpfC and RpfG protein genes derived from bacteria may cause disorder in the metabolic pathways of Xanthomonas. But when we use the same way on Xanthomonas, the pathways all act on the cell's second messenger——c-di-CMP, making it possible for us to use vc2 riboswitch and not to add the additional expression channel of other RpfC and RpfG protein. Therefore, our system is capable of adapting to all kinds of bacteria that use c-di-CMP as the second messenger. This design also allows us to save time by reducing the possible impact of gene editing on Xanthomonas. We will write in detail about the selection of engineering bacteria in 2.b. We use the Enhanced yellow fluorescent protein instead of Green fluorescent protein because, the Xoo bacterial has a strong inference in green fluorescent wave band, which could decrease the expression of GFP remarkability.
The genes of the DSF system we designed will be constructed on the pBBR plasmid, a mult host and high copy expression plasmid that can survive and express its genes in E. coli and Xanthomonas. By learning about the overexpression of protein in Xanthomonas oryzae, we know that pBBR can express higher and better expression in Xanthomonas than the specific Xanthomonas plasmid pHM. The phenomenon of protein characterization is also more obvious. Also considering the characteristics of mult host and high copy, we should reduce the possible interference in Xanthomonas, so we will also use the pBBR plasmid for experiments in the subsequent element design and testing. 2. Phage carrying system (dcas12a inhibition): 1. Purpose
After we found the elements that can control the outbreak conditions against Xanthomonas, we studied how to extend the time span of phages and our therapy. The first idea is modifying the phage to make the phage dormant in the external non-host environment to extend its lifespan. The second idea is to use bacteria to carry the phage gene, express and release it at a suitable time. However, the method of modifying bacteriophages requires too much laboratory and experimental skills, which is too much for a high school team. Also, the protein information of the bacteriophage xoo-sp2 of Xanthomonas oryzae is only partially analyzed on NCBI, and more than 70% are uncertain protein fragments, making it difficult for us to design and operate on the phage. Thus, considering these above, we chose the second option. This plan allows the phage or its genetic information to exist in the plant for a long time and be carried by bacteria.
2. Selecting detoxified Xanthomonas as the phage carrying bacteria:

When the engineered bacteria and phages were screened for carrying bacteria, we proposed the following solutions, but they all have their own advantages and disadvantages:

1. Using model bacteria represented by E. coli as engineering bacteria:

1. Experiments are easier; few interference from reporter genes and design pathways.

2. More choices for plasmids, easier to control protein expression by building mutiplasmid system.

3. Safer. Classified as Class four bacteria in the Chinese laboratory strain safety standard (the most harmless).

1. E. coli or other model strains are still not dominant strains in the natural environment and require a lot of nutritional support to maintain the population and activity.

2. Escherichia coli cannot enter the plant voluntarily, where the Xanthomonas outbreak occurs.

3. Need to add special genes to control the release of phages.

2. Using detoxified Xanthomonas:

1. Can enter the plant's ducts autonomously through the wounds of the epidermal cell and stays.

2. Easier to obtain sufficient energy and maintain activity in the plant body to survive through the energy obtained by the plant.

3. When the phage gene is expressed, the endolysin that inside Xanthomonas will automatically decompose the Xanthomonas, thereby releasing the phage.

1. As a non-model bacteria, it is necessary to acquire the component of new medium and method of bacteria culture, which increases the uncertainty.

2. Plasmids like pH2 plasmids are difficult to obtain and expensive.

3. It has the risk of infecting and killing plants.

4. Electroporation is needed to transfer the plasmid into Xanthomonas.

Considering the actual production process, we decided to use Xanthomonas as the engineering bacteria.

We decided to use two different Xanthomonas species, Xanthomonas Orzyae and Xanthomonas campestris. Xanthomonas Orzyae is in a more standardized form for engineering. Xanthomonas campestris is safer and has more experimental data. It also has lower toxicity to plants. So if we can prove that all the pathways designed can be implemented in Xanthomonas campestris, we will get a better engineered bacteria..
3. Use and selection of the CRISPR-Cas system

The next problem is how to inhibit and retain phage genes in engineered bacteria. The easiest way is to synthesize the entire piece of gene information, then import it into the pBBR plasmid. We can edit the plasmid or DNA information to control the role of DNA or RNA polymerase on this piece of DNA to achieve the effect of control and release. However, the length of the information about Xanthomonas oryzae xoo-sp2 that we found on ncbi reached 60,000. If the whole gene synthesis is used, the cost will be expensive. There are also many uncertainties in segmented synthesis. Because there are more than 50 percent hypothetical proteins in the 150 parsed proteins information, we cannot know the exact function of many proteins. Therefore, we decided not to introduce the entire gene of the phage.

In the mutual evolution of phages and their host bacteria, bacteria developed the CRISPR-Cas system and phages developed the Anti-CRIPRS protein. Since Xanthomonas and its phages do not have such an inhibitory system, we decided to use the dCas12a system from Francisella novicida to inhibit the expression of Xanthomonas phage genes in bacteria.

Figure4: dcas12a design
4. Select endolysin and major tail as the target protein on the phage:

After we decided to use dcas12a, we investigated and studied all known proteins of xoo-sp2. Among them, we believe that when the phage infects the host bacteria, the inhibition of two key proteins, tail protein and endolysin, can well retain the phage gene in the engineered bacteria. Xanthomonas phages have tail protein that can enter and infect host bacteria. Phages produce offspring by injecting tail protein into cells and using the host bacteria's RNA and DNA polymerase. Xanthomonas phage also has endolysin protein, a water-soluble protein that can break the chemical bonds between the cell walls of host cells. Endolysin generally accumulates in large amounts in the final stage of phage infection, causing the phage to be released from Xanthomonas.

5. Design of sgRNA:

SgRNA is the single guide RNA. As one of the two components of a simple CRISPR nuclease system. Formed by combining crRNA and trRNA into a single synthetic guide RNA. SgRNA can be transferred as RNA or transformed by plasmids with sgRNA coding sequence under the promoter.

When designing sgRNA, we used the design software and algorithm on the CHOPCHOP website, imported endolysin (BBa_K3820001) and major tail protein (BBa_K3820007) respectively. We chose the sgRNA with the highest score on the website as the sgRNA that we expressed on the plasmid: (BBa_K3820002) (BBa_K3820003)

Figure5: phage carrying system design
6. plasmid design:

The Xanthomonas engineering bacteria that we selected will transformed the dcas12a expression pathway through electrotransformation, and target the sgRNA expression pathways of endolysin and major tail protein in Xanthomonas phages, major tail protein (BBa_K3820026) and endolysin (BBa_K3820025) expression pathway. We plan to use IPTG to induce and regulate the expression of dcas12a (BBa_K3820022), and use cumate to induce and regulate the expression of sgRNA (BBa_K3820023). In such a design, we can observe whether the protein is expressed or not through protein gel electrophoresis and some apparent phenomena of bacteria.
3. Phage release system:
1. Purpose:

This system is designed to release phage at the appropriate time. This system needs to play the role of switch, which can turn off the phage carrier system and activate phage expression.

2. AcrVA1

Anti-CRISPR protein(AcrVA1), as a phage protein that specifically targets the bacterial CRISPR protein, can attack the CRIPSR protein through different targets. We found the AcrVA1 protein in the V. cholera phage can target the dcas12a protein in V. cholerawe used in the carrying system. When AcrVA1 protein is expressed, Cas12a-AcrVA1 complex form and binds to Cas12a independent on crRNA. On the bind site of Cas12a protein, it carries the positive charge, and complementary to the overall negatively charge of AcrVA1.
We plan to regulate AcrVA1 through the DSF sensor system, so that when the bacteria enter the rapid growing phase, the Acr protein will be expressed and the effect of the dcas12a protein will be reduced. Plentiful of phages will grow in the bacteria subsequently.
Figure6: AcrVAI protein structure design
3. Plasmid design:

We built the AcrVA1 element on a conventional protein expression pathway. We also decide to set up a control group by transferring plasmids and non-transferring plasmids.
4. GdpX1 overexpression system: 1. Purpose:

Since the engineered bacteria, Xanthomonas campestris and Xanthomonas oryzae, are both toxic to plants. In order to prevent plants from being affected by our products, we have investigated how Xanthomonas attacks plants and how Xanthomonas breeds the offspring. Besides, we discovered that the T3SS secretion system in Xanthomonas has the function of secreting toxins, reducing the activity of the plant's immune system and improving its own exercise capacity. However, these two effects should not occur in the genetic engineered Xanthomonas. Because the T3SS secretion system in Xanthomonas is complicated, the coercive interference of this secretion system through synthetic biological may lead to the death of the bacteria. Taking the aforementioned discussion into account, we found a self-stopping mechanism in Xanthomonas, which is GGDEF-domain protein (GdpX1). Through literature investigation, GdpX1 is a protein present in Xanthomonas oryzae PXO99A strain. In the paper, by knocking out the GdpX1 protein on Xanthomonas, they discover that virulence of Xanthomonas, exopolysaccharide production, and flagellar motility increase significantly. Besides, after overexpression of GdpX1 protein in Xanthomonas, the toxicity of Xanthomonas is reduced significantly. The GdpX1 protein basically conforms to our hypothesis for low toxicity Xanthomonas. Therefore, we will transfer the gene sequence pathway that overexpresses GdpX1 in the engineered Xanthomonas.

Figure7: AcrVAI design
2. plasmid design:

We use the method of IPTG induction, by setting up the control groups with different expression levels of Xanthomonas, we observe and confirm the effect of overexpression of GdpX1 on Xanthomonas through plant and microscope experiments.

Figure8: GdpX1 design

Figure9: Pathways overview
5.Overall description: Without the DSF molecule, Vc2 riboswitch binds to c-di-GMP, and the downstream Acr protein cannot be expressed. Dcas12a and sgRNA will continue to be expressed by combining with the expression pathway of Xanthomonas phages major tail protein and endolysin. Phage won't be released because neither endolysin nor major tail protein can be expressed normally.

With the DSF molecules, the connection between Vc2 riboswitch and c-di-GMP is broken, and the downstream Acr protein is expressed. AcrVA1 protein acts on the binding site of sgRNA, shortening the binding site and releasing the expression pathway of endolysin and major tail protein. The phage grows within the engineered Xanthomonas rapidly, before the engineered Xanthomonas is decomposed. Finally the phage enters the external environment and dispose the invasive Xanthomonas.
Reference: 1. Zhang, F., Song, G., & Tian, Y. (2019). Anti‐CRISPRs: The natural inhibitors for CRISPR‐Cas systems. Animal Models and Experimental Medicine.

2. Dong, Z., Xing, S., Liu, J., Tang, X., Ruan, L., Sun, M., … Peng, D. (2018). Isolation and characterization of a novel phage Xoo-sp2 that infects Xanthomonas oryzae pv. oryzae. Journal of General Virology, 99(10), 1453–1462.

3. Miao, C., Zhao, H., Qian, L., & Lou, C. (2019). Systematically investigating the key features of the DNase deactivated Cpf1 for tunable transcription regulation in prokaryotic cells. Synthetic and Systems Biotechnology, 4(1), 1–9.

4. Yang, F., Qian, S., Tian, F., Chen, H., Hutchins, W., Yang, C.-H. ., & He, C. (2016). The GGDEF-domain protein GdpX1 attenuates motility, exopolysaccharide production and virulence in Xanthomonas oryzae pv. oryzae. Journal of Applied Microbiology, 120(6), 1646–1657.

5. Smith, K. D., & Strobel, S. A. (2011). Interactions of the c-di-GMP riboswitch with its second messenger ligand. Biochemical Society Transactions, 39(2), 647–651.