Potassium as a Macro-nutrient

Potassium is an essential plant macronutrient as it has numerous important roles in plants including osmoregulation, CO₂ regulation, starch 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 kdpFp, a promoter located upstream of KdpFABC operon in Escherichia coli (E. coli) which works under low [K⁺] condition. We put it upstream of a GFP reporter so as to characterize the promoter activity.

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Endogenous K sensing system

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The potassium ion uptake in E. 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?). kdpFABC operon is 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. KdpD is stimulated by both intracellular and extracellular potassium ion concentration (Jung ,2000; Jung, 2001; Roe, 2000; Yan, 2011a; Laermann, 2013). The autophosphorylation of KdpD transfers a phosphoryl group to the KdpE upon low potassium concentration (Voelkner, 1993; Puppe, 1996; Jung ,1997a; Jung, 2000). Under an increase in potassium ion concentration, KdpD phosphatase activity will be enhanced, causing a decrease in phospho-KdpE and kdpFABC expression. Phosphorylated KdpE activates kdpFABC operon (Zhang, 2014a; Laermann, 2013).

The kdpFp we adopted is upstream of the kdpFABC operon with -28 position of transcription start site relative to start the first gene, kdpF. The -10 and -35 box elements of have been mapped are TACCCT and TTGCGA respectively (Sugiura et al., 1992). The transcription factor, phosphorylated KdpE, binds to the kdpFp at binding site located from -71 to -55 site with reference to the transcription start site to initiate the transcription of downstream gene (Sugiura et al., 1992; Narayanan et al., 2012).

Design of K⁺ sensing Device

As our potassium-sensing device, we obtained the promoter, kdpFp, and then combined it with a downstream GFP generator, BBa_E0240, using BioBrick RFC 10 standard so that the promoter activity in different potassium level can be detected and characterized.

EcoRI Illegal Site

In order to make our promoters, kdpFp, compatible with Biobrick RFC 10 standard and so that readily accessible to the whole iGEM community, we have to remove the EcoRI illegal site from -18 to -12 position within. We removed the illegal site by mutating the thymine at -15 position with reference to the transcription start site to guanine, cytosine and adenine to give rise to 3 promoter mutants. We expected that the four promoters would differ in activity because of the difference in binding energy between the promoter and RNA polymerase due to the base-pair changes (Brewster, 2012). Therefore, we characterized all of the promoters to compare their strengths by means of their respective fluorescence levels and RPU measurements so as to obtain a more comprehensive knowledge in the activity and working range of the four promoters. For convenience, we denote them as kdpFp(A)/A-mutant, kdpFp(G)/G-mutant and kdpFp(C)/C-mutant respectively in the following context.

Interference from other Endogenous Systems

Another concern is the masking of kdpFp activity by other native constitutive potassium transport systems in E.coli. Other than the inducible KdpFABC system(K_M= 2 µM), E. coli has Trk and Kup systems which are constitutively expressed, low-affinity potassium transport systems (K_M= 1.5 mM) (reference?). A study has found that the expression level of KdpFABC is generally higher in a strain without Trk and Kup system (Laimins et al., 1981). It is due to the fact that E. coli uses the two saturable transporters, Trk and Kup, under normal physiological conditions to uptake extracellular potassium ion. Only at a very low concentration of potassium ion, that these two systems can no longer satisfy the need of potassium ion of E. coli, the kdpFp will be activated and drive the expression of KdpFABC complex to uptake more K⁺. As the result, the activity of kdpFp may be masked by Trk and Kup system and cannot be truly reflected (Laermann et al., 2013). We decided to measure the activity of kdpFp in E.coli TK2240 (kdp+ Δtrk Δkup) strain, which is a strain defectve in Trk and Kup system. Such that we are able to characterize kdpFp promoter in a more accurate manner.

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). Additionally, we intended to find the comparison of the activities between different promoters, thus, we also measured Relative Fluorescence Unit (RFU) of the four potassium promoters.

Relative promoter unit measurement

badass rpu graph

Figure . Relative promoter unit (RPU) of kdpFp[-15,T>G] across different concentration of K⁺. The measurement of the activity of G mutant was carried out using fluorescence-activated cell sorting. The strength of the promoter is presented in relative to the strength of standard reference promoter. Error bar are represented as SEM.

From the lowest concentration of potassium ion to 0.025 mM, the strength of the G mutant promoter was found to be about 0.5 RPU and decreasing when concentration increased. After the concentration of potassium ion at 0.025 mM, there was no significant change on the RPU values. Strength of G mutant kdpFp at the lowest concentration was about 1.5 times higher than those after 0.025 mM.

High concentration of potassium ion could repress the expression of kdpFp, due to satisfaction of potassium ion by constitutively expressed low-affinity K⁺ transporter system, Trk and Kup(Laermann et al., 2013). Therefore the activity of kdpFp promoter, that is represented by the relative fluorescence intensity, at high concentration of potassium ion, should be low and it is shown in our experiments.

Relative fluorescence measurement

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PAIRED graph to be put here

Figure . Activity of kdpFp in E. coli DH10B in different K⁺ concentrations. Pair graph representing the activity of different kdpFp. Fluorescence/absorbance versus [K⁺] plot is shown on the left while the GFP synthesis rate versus [K⁺] plot is on the right. A, T(wild type), C, and G represent A mutant, wild type promoter, C mutant and G mutant respectively. Error bar are represented as SEM.

Both C and G mutants similarly expressed higher fluorescence intensity compared to the wild type and the A mutant. A higher level of potassium ion inside K minimal medium repressed the expression level of kdpFp, which was shown by lower fluorescence intensity of all promoters at higher concentration of potassium ion. The expression level of both C and G mutant promoters were significantly higher than the wild type one, while A mutant was always the lowest.

The fluorescence intensity decreases over the concentration of potassium ion in K minimal medium. After several trials, we found the dynamic range of our promoters. It is between 0 to 0.1 mM of potassium ion in K minimal medium. Thus, we characterized our promoters at those range of concentrations (0 to 0.2 mM).

We observed that the fluorescence intensity expressed by C and G mutants are always at around similar values since the binding affinity might be the same with reference to the energy matrix (reference?). Moreover, both of them are always higher than the one expressed by the wild type promoter. Meanwhile, the fluorescence intensity expressed by A mutant is lower than the the one expressed by the wild-type promoter. Also, the relationship of relative fluorescence intensity and K⁺ concentration of all the promoters are coherent with the previous RPU measurement of G mutant.

Figure . The activities of kdpFp in the medium containing no potassium ion.A fluorescence/absorbance plot was obtained from measuring the relative fluorescence level exhibited by the promoters, kdpFp and its 3 mutants (A, C and G), in DH10B cells in 0 mM K minimal medium. The expression level of both C and G mutants are significantly higher than the expression level of wild-type promoter and A mutant promoter. There was no significant difference between the activity of G mutant and C mutant promoters, as well as between wild type promoter and A mutant. Error bars are represented as SEM.

We also would like to make a clear comparison between the expression level of the different mutant promoters. We tried to make a comparison of the expression of all the promoters at the lowest level of potassium ion, that is no potassium ion in K minimal medium. From the graph that we obtained, we observed that the expression level of both C and G mutants are about 2 times higher than the wild type promoter and A mutant. These results demonstrate our former experiments that the relative fluorescence intensity level of both C and G mutants are always higher than both wild type promoter and A mutant.

Figure . Comparison between the activities of kdpFp[-15, T>G] in DH10B and TK2240 strain. A fluorescence/absorbance plot was obtained from measuring the relative fluorescence level exhibited by the G mutant pormoter in DH10B and TK2240 cells. At the concentration of potassium ion lower than 0.0125 mM, the fluorescence intensity expressed by G mutant in DH10B was significantly greater than the one in TK2240 strain. The fluorescence intensity expressed by G mutant in DH10B strain decreased over the concentration, while the one in TK2240 remained the same. Starting from the concentration of potassium ion at 0.0125 mM onwards, the difference of GFP expressed by both strains was not significant. After passing the concentration of potassium ion at 0.05 mM, the expression level of kdpFp in TK2240 strain exceeded the one in the DH10B strain. Error bars are represented as SEM.

We did another relative fluorescence measurement using TK2240 to observe a more accurate activity of kdpFp. The TK2240 strain is defective in trk and kup gene. The only gene which could respond to potassium ion in TK2240 strain is only kdp gene. Therefore the expression of kdp gene would not be masked by the saturable low-affinity K⁺ transporter system. We were expecting that the activity of kdpFp in TK2240 are higher than that in DH10B. The fact mentioned earlier corresponds to the graph at higher concentration of potassium ion, although the expression level in DH10B is higher than in TK2240 at low concentration of potassium ion.

We could observe the difference of the fluorescence intensity expressed by both strains at the concentration of potassium ion lower than 0.0125 mM. The fluorescence intensity expressed by G mutant in DH10B strain decreases over the concentration, while the one in TK2240 remains the same. Since TK2240 has a greater need of KdpFABC complex to help scavenge the external K⁺, so what might have caused this would be TK2240 spend too much energy at that stage to make KdpFABC complex and thus spare less energy on producing GFP.

At higher concentration of potassium ion, starting from 0.0125 mM onwards, the difference of fluorescence intensity expressed by both strains is not significant anymore. Both strains show decrease in fluorescence intensity over the increasing concentration of potassium ion in K minimal medium.

After passing the concentration of potassium ion at 0.05 mM, the fluorescence intensity expressed by TK2240 strain exceeds the one in the DH10B strain due to the constant need of potassium ion of the cells and in TK2240, there is only KdpFABC transporter system that can satisfy this need of E. coli.

Considerations for replicating the experiments

a. OD₆₀₀= 0.4 referring to mid log phase of E. coli

b. Dilution has always been done to make the OD600 values of start culture around the same before subculturing in different concentration of K⁺.