Team:Nanjing high school/Design
Design
Overview
Phosphate solubilizing microbes have the potential to promote phosphorus absorption by crops and reduce the use of phosphate fertilizers. Tatumella citrea, which belongs to this kind of microbe provides phosphorus for crops (Di-Simine et al. 1998; Bar-Yosef et al. 1999). To build up a regulable genetic circuit, we chose one of its essential gene GAPDH and made it the substrate regulated by salicylic acid, which can be secreted by crops rhizospheres. We set up a bio-sensor system made of nahR gene and PsaI promoter, and we chose EGFP to verify if this bio-sensor worked well.
Expectation
-The expectation of bio-sensor
The bio-sensor (nahR gene and Psal promoter) for the detection of salicylic acid we adopted is originally on the chromosome of Pseudomonas Stutzeri (Bosch, Rafael, et al.). With the appearance of salicylic acid, the protein translated form nahR binds with PsaI promoter and initiates the expression of the enzyme that can utilize the acid as one of the energy sources for P. Stutzeri. In our design, we replaced the enzyme with an essential gene for the metabolism of T. Citrea (Whitelaw 2000; Maliha et al. 2004). Therefore, it can only live around the roots that produce salicylic acid.
To achieve our goal of setting up a regulable genetic circuit of phosphate solubilizing, we expected to build up a bio-sensor system by using EGFP as a reporter in T. citrea and when salicylic acid was added in the culture, we can detect the expression of EGFP. In the first phase of our experiment we will replace the essential gene with the GFP gene in our design to test our framework due to the complexity of removing essential gene of T. Citrea. With GFP, the ideal situation is that the edited bacteria will be fluorescent with the existence of salicylic acid. Through adding up different concentration of salicylic acid in the culture of this strain, we were looking forward to detecting different amount of fluorescence intensity. After measuring the dosage of the salicylic acid, we were able to measure the time course of its valid time. If this bio-sensor system works well in T. citrea, we will be able to set up a further use by constructing a GAPDH knock-out strain and restricting this phosphate solubilizing bacteria to the rhizosphere of plants to increase the use of phosphorus in the soil.
Process
For our project, we need to ensure the regulable bio-sensor works well and to know if it can be regulated by salicylic acid. So that we can make sure the GAPDH-knocked-out T. citrea can commensalism with crops. Therefore, the efficiency of phosphate solubilizing can be promoted and decrease the phosphorus pollution.
Principle explaination
The transcription factor NahR found in Pseudomonas putida can specifically bind to salicylic acid to actiavate the Psal promoter. According to this, our regulable genetic circuit is designed to control the expression of the essential gene GADPH of T. citrea through PsaI. So that in the presence of salicylic acid, GADPH can be expressed, and T. citrea can survive. Since salicylic acid is a root exudate, this genetic circuit that T. citrea can commensalism with crops.
How this works: the expression of nahR gene → add salicylic acid in the culture → EGFP/GAPDH gene expression → detect the fluorescence →bio-sensor works well→T. citrea can commensalism with crops
Cores
In our project, we learned how to use molecules to regulate the processes in synthesis biology and how to construct plasmids. We also learned how to use Fluorescence microscope to detect EGFP, and we used Spectra Max Plus 384 to measure both the fluorescence intensity and OD600 of the strains.
Citation
Bar-Yosef, B., et al. “Pseudomonas Cepacia-Mediated Rock Phosphate Solubilization in Kaolinite and Montmorillonite Suspensions.” Soil Science Society of America Journal, vol. 63, no. 6, 1999, pp. 1703–1708., doi:10.2136/sssaj1999.6361703x.
Bosch, Rafael, et al. “Complete Nucleotide Sequence and Evolutionary Significance of a Chromosomally Encoded Naphthalene-Degradation Lower Pathway from Pseudomonas Stutzeri AN10.” Gene, vol. 245, no. 1, 2000, pp. 65–74., doi:10.1016/s0378-1119(00)00038-x.
Maliha Rashid, et al. “Organic Acids Production and Phosphate Solubilization by Phosphate Solubilizing Microorganisms (PSM) Under in Vitro Conditions.” Pakistan Journal of Biological Sciences, vol. 7, no. 2, 2004, pp. 187–196., doi:10.3923/pjbs.2004.187.196.
Whitelaw, M.a. “Growth Promotion of Plants Inoculated with Phosphate-Solubilizing Fungi.” Advances in Agronomy, 1999, pp. 99–151., doi:10.1016/s0065-2113(08)60948-7.
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