Revision as of 03:15, 30 August 2015

Potassium Sensor

Potassium as a Macro-nutrient

Potassium is an essential plant macronutrients as it has numerous roles in plants and it is required for plant growth and development. Some of its important roles include the regulation of opening and closing of stomata which therefore regulates water (osmoregulation) and CO₂. It is also essential in starch synthesis and protein synthesis. Moreover, it activates many growth related enzymes in plants. Hence, the deficiency of K+ ion will result in abnormalities in plant growth and metabolism.

What we try to achieve

On account of crucial impact that K⁺ can contribute to the plant performance, 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.

Potassium sensor Design

We utilized kdpFp, a promoter located upstream of kdpFABC operon in Escherichia coli (E. coli) which works under low K⁺ concentration in pursuance of a precise and functional Potassium Sensor. However, there is an illegal EcoRI site in KdpFp promoter, thus, we attempted to remove that illegal site by constructing G-Mutant kdpFp, C-Mutant kdpFp and A-Mutant kdpFp. Those 3 promoters have the illegal site removed by G (guanine), C (Cytosine) and A (Adenine).

Measurement and Characterization

Upon different concentration of K⁺, Potassium sensor will show different fluorescence level due to distinctive effect of K⁺ ion to kdpFp. We characterized kdpFp by using RPU as standard unit. The measurement results were obtained with FACS (Fluorescence-activated cell sorting) and EnVision multilabel reader.

Achievement

Hitherto, our team has finished characterizing all the constructs and contemplated the activity of the promoters over a varying range of K⁺ concentration.

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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 					<hr class="para">
 					<h1>Mechanism</h1>
-					<p>In our project, we use the native potassium ion transport system in <i>Escherichia coli</i> (<i>E. coli</i>), Kdp system as our potassium sensing part. The Kdp system is composed of two major parts, <i>KdpFABC</i>, a high-affinity potassium transporter as well as two types of regulatory proteins, a sensory kinase <i>KdpD</i> and a response regulator <i>KdpE</i>. <i>KdpD</i> and <i>KdpE</i> works together as a two-component system, tracking and responding to the intra and extracellular potassium level then interacting with the <i>KdpFABC</i> encoding operon. <i>kdpFABC</i> operon is up-regulated under low potassium ion concentration and is inhibited under high concentration. <br><br>
+					<p>In our project, we use the native potassium ion transport system in <i>Escherichia coli</i> (<i>E. coli</i>), 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 <i>KdpD</i> and a response regulator <i>KdpE</i>. <i>KdpD</i> and <i>KdpE</i> works together as a two-component system, tracking and responding to the intra and extracellular potassium level then interacting with the <i>KdpFABC</i> encoding operon. <i>kdpFABC</i> operon is up-regulated under low potassium ion concentration and is inhibited under high concentration. <br><br>
 <i>KdpD</i>, which is a trans-membrane protein, auto-phosphorylates itself, also phosphorylates and dephosphorylates <i>KdpE</i>. Low concentration of potassium ions favors the phosphorylation of <i>KdpE</i>, which then gives rise to the enhancement of the level of phosphorylated <i>KdpE</i>, and as a result, triggers the up-regulation of <i>kdpFABC</i> operon.<br><br>

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