Team:HKUST-Rice/Description

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Overview

Nitrogen (N), Phosphorus (P), and potassium (K) are three macronutrients for plants, 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 is constructing a biological sensor in E. coli, which can detect NPK levels in the surrounding environment and give responses in the form of colors. In addition, we are characterizing 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.

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+ concentration (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 concentration of K+ and express different level of GFP accordingly.

PphoA promoter (Hsieh, Y. J., & Wanner, B. L.,2010).is cross-regulated by phoB and phoR, and is usually repressed under high phosphate concentration. 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 activates the expression of PphoA promoter.

For nitrate sensing, we decided to use PyeaR promoter (Lin, et al, 2007). It is normally cross-regulated by the Nar two-component regulatory system (T.Nohno, et,al. , 1989) and nsrR, a regulatory protein that prevents the transcription of a number of operons in E. coli. When there is nitrate and nitrite in the environment, it will enter the cell and then being converted into nitric oxide. The nitric oxide will bind to nsrR and relieve the repression on the PyeaR promoter. As a result, any genes that are downstream of the PyeaR promoter will be expressed. As the biobrick of PyeaR promoter with a GFP coding device has already been submitted by the University of Bristol in 2010, we could characterize it and directly apply to our biosensor.

In order to characterize the output difference between a single expression system and co-expression from a one vector, we will be constructing 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 aim 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 (N, P and K) promoters.

Reference:

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.