Project

Overview

Potassium (K), phosphorus (P), and nitrogen (N) are three plant macronutrients, and deficiencies in any of these can lead to plant diseases. By creating a biological sensor that can quickly provide soil status to plant owners, we can prevent plant diseases due to the lack of nutrients. In view of this, our team constructed a biological sensor in E. coli, which can detect KPN levels in the surrounding environment and give responses in the form of colors. In addition, we characterized the effects of a dual output system, in contrast to a single output system, in order to anticipate the expression of multiple outputs in a single system.

Potassium Sensor

Phosphate Sensor

Nitrate Sensor

KdpFABC transporter is a high affinity K⁺ uptake system (Siebers, A. & Altendorf, K., 1988). The promoter upstream of kdpFABC operon, kdpFp, works under low K⁺ concentrations (Polarek, J. W., et al., 1992; Walderhaug, M. O., et al., 1992). The goal is to characterize kdpFp and build a device which is able to sense different concentrations of K⁺ and express different levels of GFP accordingly.

phoAp and phoBRp promoters (Hsieh, Y. J., & Wanner, B. L., 2010) are cross-regulated by phoB and phoR, and are usually repressed under high phosphate concentrations. phoR behaves as an activator as well as an inactivator for phoB. When phosphate is limited, phoR will phosphorylate phoB and the phosphorylated phoB will directly activate the phoAp and phoBRp promoters.

yeaRp promoter (Lin, et al., 2007) is normally cross-regulated by the Nar two-component regulatory system (T. Nohno, et al. , 1989) and NsrR, a regulatory protein. When there is nitrate and nitrite, it will be converted into nitric oxide. The nitric oxide will bind to NsrR and relieve the repression on the yeaRp promoter. As a result, any genes that are downstream of the yeaRp promoter will be expressed.

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Signal Coexpression

In order to characterize the output difference between a single expression system and co-expression from one vector, weconstructed several inducible systems that give off fluorescence output and compare the dose response of the individual systems in the two scenarios.

As part of the expression platform, we also aimed to construct a 3-input AND logic gate using toehold switches (Green, A. A., et al., 2014) to integrate the input signals detected from the three (K, P, and N) promoters.

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References:

Nohno, T., Noji, S., Taniguchi, S., & Saito, T. (1989). The narX and narL genes encoding the nitrate-sensing regulators of Escherichia coli are homologous to a family of prokaryotic two-component regulatory genes. Nucleic acids research,17(8), 2947-2957.

Lin, H. Y., Bledsoe, P. J., & Stewart, V. (2007). Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen-responsive regulator Fnr in Escherichia coli K-12. Journal of bacteriology, 189(21), 7539-7548.

Hsieh, Y. J., & Wanner, B. L. (2010). Global regulation by the seven-component P i signaling system. Current opinion in microbiology, 13(2), 198-203.

Siebers, A. and Altendorf, K. (1988). The K+-translocating Kdp-ATPase from Escherichia coli. European Journal of Biochemistry, 178, 131–140.

Walderhaug, M. O., Polarek, J. W., Voelkner, P., Daniel, J. M., Hesse, J. E., Altendorf, K., & Epstein, W. (1992). KdpD and KdpE, proteins that control expression of the kdpABC operon, are members of the two-component sensor-effector class of regulators. Journal of Bacteriology, 174(7), 2152–2159.

Green, A.A., Silver, P.A., Collins, J.J., & Yin, P. (2014). Toehold Switches: De-Novo-Designed Regulators of Gene Expression. Cell, 157(4), 925-935.