Team:HKUST-Rice/Potassium Sensor
Potassium Sensor - kdpFp
Potassium as a Macro-nutrient
Potassium is an essential plant macronutrients as it has numerous important roles in plants including osmoregulation, CO2 regulation, starch synthesis and protein synthesis. Hence, the deficiency of K+ ion will result in abnormalities in plant growth and metabolism. It is fundamental to determine its concentration in soil in order to provide the proper amount of additional potassium that must be added to the plant by any particular fertilizers.
Our aim is to engineer a Potassium sensor that can detect a range of K+ concentration in the soil to ensure the suitable soil condition for the plant fitness. We utilized PKdpF, a promoter located upstream of KdpFABC operon in Escherichia coli which works under low [K+] condition. We put it upstream of a GFP reporter so as to characterize the promoter activity. For simplification, PKdpF will be called potassium promoter in the following context. There is an illegal EcoRI site in PKdpF promoter. To make the potassium promoter readily accessible to the whole iGEM community, we removed that illegal site by mutating the thymine at -15 position to guanine, cytosine and adenine to give rise to 3 promoter mutants.
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Endogenous K sensing system
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The potassium ion uptake in Escherichia coli is regulated by several systems under different conditions. The potassium ion transporters, Trk and Kup are constitutively expressed (reference?) while KdpFABC, another transporter is activated under limited potassium ion concentration (reference?) and controlled by the KdpDE two-component system (TCS) (Polarek, 1992; Walderhaug, 1992). The TCS consists of KdpD which is a membrane-bound sensor kinase and KdpE which is the cytoplasmic response regulator. The autophosphorylation of KdpD transfers a phosporyl group to the KdpE upon low potassium concentration (Voelkner, 1993; Puppe, 1996; Jung ,1997a; Jung, 2000). Phosphorylated KdpE activates kdpFABC operon.
KdpDE TCS is stimulated by both intracellular and extracellular potassium ion concentration (Jung ,2000; Jung, 2001; Roe, 2000; Yan, 2011a; Laermann, 2013). The intracellular and extracellular potassium ion concentration shows an inverse correlation to KdpDE phosphorylation. Under an increase in potassium ion concentration, KdpD phosphatase activity will be enhanced, causing a decrease in phospho-KdpE and kdpFABC expression. Decreasing potassium ion concentration shows the other way around (Zhang, 2014a; Laermann, 2013).
KdpFABC transporter, which depends on ATP, is a high affinity P-Type ATPase potassium ion uptake system (Siebers, 1988; Siebers 1989). The KdpFABC transporter is specific to potassium ion (Km= 10 μM), magnesium ion (Km= 80 μM), as well as other particular substrates (Siebers, 1988). The KdpFABC complex consists of kdpA, kdpB, kdpC, and kdpF subunits. The kdpA subunit controls the binding as well as transportation of substrate and kdpB subunit pairs the energy. The kdpC subunit keeps kdpA and kdpB together (Buurman95). In addition, the kdpF gene encodes a small polypeptide which stabilize the KdpFABC complex (Altendorf98, Gassel99).
Experimental design
Measurement and Characterization
In order to assemble a device that can be widely used by all iGEM community, we characterized kdpFp promoter by using Relative Promoter Unit (RPU) as standard unit. RPU is the standardized measurement unit that use BBa_J23101 (constitutive promoter) to serve as an in vivo reference standard for promoter activity. Consequently, our constructs can be easily utilized and reproduced by any other iGEM participants. The measurement for RPU was obtained with FACS (Fluorescence-activated cell sorting). Additionally, we also intended to find the comparison of the activities between different promoters, thus, we also did Relative Fluorescence Unit (RFU) measurement. From this RFU measurement, we processed the data and obtained the Green Fluorescence Protein (GFP) synthesis rate between those different promoters. As a result, we could choose the optimum promoter to be used as our Potassium sensor.
Achievement
Our team has finished characterizing all the constructs, including the wild type promoter and 3 mutants controlling the expression of GFP. We also contemplated the activity of the promoters over a varying range of K+ concentration. We had discovered a comparison between different promoters and a dynamic relationship between K+ concentration and the promoters’ activity. Upon different concentration of K+, Potassium sensor will show different fluorescence level due to distinctive effect of K+ ion to kdpFp.
Mechanism
In our project, we use the native potassium ion transport system in Escherichia coli (E. coli), Kdp system as our potassium sensing part. The Kdp system is composed of two major parts, KdpFABC, a high-affinity potassium transporter as well as two types of regulatory proteins, a sensory kinase KdpD and a response regulator KdpE. KdpD and KdpE works together as a two-component system, tracking and responding to the intra and extracellular potassium level then interacting with the KdpFABC encoding operon. kdpFABC operon is up-regulated under low potassium ion concentration and is inhibited under high concentration.
KdpD, which is a trans-membrane protein, auto-phosphorylates itself, also phosphorylates and dephosphorylates KdpE. Low concentration of potassium ions favors the phosphorylation of KdpE, which then gives rise to the enhancement of the level of phosphorylated KdpE, and as a result, triggers the up-regulation of kdpFABC operon.
As our potassium-sensing device, we adopt the promoter kdpFp from kdpFABC operon. The sequence was obtained by oligos, we then combine the promoter with the downstream GPF generator using biobrick RFC 10 so that the change of the promoter activity in different potassium level can be detected and characterized.
Limitations
There are two major limitations in making use of the Kdp system as our potassium-sensing module. The first main concern is that the promoter kdpFp contains the EcoRI illegal site. While the second concern is about the background noise contributed by other native constitutive potassium transport systems of E. coli, including trk and Kup systems, which are potassium ions influx systems and are expected to lower the activity of our promoter kdpFp.
Solutions to the limitations
We have come up with solutions to tackle the aforementioned limitations of Kdp system. For the EcoRI illegal site inside the promoter, we ordered 4 different versions of kdpFp, one of them is the wild-type promoter; for the other three, they have one base-pair at -15 site, where the illegal site locate, changes from thymine (T) to cytosine (C), guanine (G) and adenine (A) respectively. This make the three promoters into three different mutants, we denote them as A-mutant, G-mutant and C-mutant respectively. All the mutants, thereby, have their illegal site removed.
Result obtained
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