Potassium sensor Design

We utilized kdpFp, a promoter located upstream of kdpFABC operon in Escherichia coli which works under low K⁺ concentration in pursuance of a precise and functional Potassium Sensor. However, there is illegal EcoRI site (GAATTC) which is located in -15 position relative to start site of transcription in kdpFp promoter, thus, we overcome that illegal site by changing thymine base in -15 position relative to transcription start site into adenine, cytosine, and guanine.

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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