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. The deficiency of K⁺ ion will result in abnormalities in plant growth and metabolism. 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. We utilized kdpFp, a promoter activated at under low [K⁺] condition. We put it upstream of a GFP reporter so as to characterize the promoter activity.

Figure 1. Engineered E. coli as a biosensor of K⁺.

Endogenous K⁺ sensing system in E. coli

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 (Epstein & Kim, 1971) while KdpFABC, another transporter is activated under low [K+] condition (Laimins et al., 1981).

Figure 2. Endogenous K⁺ uptake systems.

The kdpFABC operon is controlled by the KdpDE two-component system (TCS) which consists of KdpD, a membrane-bound sensor kinase, and KdpE, a cytoplasmic response regulator (Polarek, 1992; Walderhaug, 1992). KdpD is stimulated by both intracellular and extracellular potassium ion concentration (Jung, 2000; Jung, 2001; Roe, 2000; Yan, 2011a; Laermann, 2013). KdpD phosphorylates 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 turns on the expression of kdpFABC (Zhang, 2014a; Laermann, 2013).

The kdpFp 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 have been mapped are TACCCT and TTGCGA respectively (Sugiura et al., 1992). The transcription factor, phosphorylated KdpE, binds to the P_kdpF 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

To make a potassium-sensing device, we obtained the promoter, kdpFp, and combined it with a GFP reporter, BBa_E0240, in BioBrick RFC10 standard so that the promoter activity in different potassium level can be detected and characterized.

Figure 3. K⁺ sensing construct with reporter.

EcoRI Illegal Site

To make our promoters compatible with RFC10 standard and, as so, readily accessible to iGEM community, we designed variants of kdpFp with EcoRI site removed. We mutated the thymine at -15 position to guanine, cytosine and adenine. The wild type promoter and the 3 variants are expected to be different in activity because of the difference in binding energy between the promoter and RNA polymerase (Brewster, 2012). Therefore, we characterized all of them to compare their strengths by relative fluorescence intensity. so as to obtain a more comprehensive knowledge in the activity and working range of the four promoters. For convenience, we denote them as A mutant, G mutant and C mutant respectively in the following context.

Interference from other Endogenous Systems

Another concern is the masking of P_kdpF activity by other native constitutive potassium transport systems in E. coli. Other than the inducible KdpFABC system, E. coli has Trk and Kup systems which are constitutively expressed, low-affinity potassium transport systems (Epstein & Kim, 1971). A study has found that the expression level of KdpFABC is generally higher in a strain without Trk and Kup system (Laermann et al., 2013). As the result, the activity of P_kdpF may be masked by Trk and Kup system and cannot be truly reflected. We decided to measure the activity of kdpFp in E. coli TK2240 (kdp+ Δtrk Δkup) strain, which is a strain defective in Trk and Kup system. Such that we are able to characterize P_kdpF promoter in a more accurate manner.

Measurement and Characterization

In order to assemble a device that can be widely used by iGEM community, we characterized the kdpFp promoter by relative promoter unit (RPU) measurement. Additionally, relative fluorescence unit (RFU) measurement has also been done to compare the activities between wild type promoters and 3 variants.

Relative promoter unit measurement

Figure 4. Relative promoter unit (RPU) of kdpFp[-15,T>G] at different concentration of K⁺. Cells were pre-cultured in K115 medium overnight at 37°C. The cells were washed 3 times in 0.85% saline and then sub-cultured in medium with specific [K⁺]. Cells were fixed when OD₆₀₀=0.4. The measurement was carried out using fluorescence-activated cell sorting. Error bar are represented as SEM.

At lowest [K⁺], the strength of the G mutant promoter was found to be about 0.5 RPU. RPU of the promoter decreased when concentration increased. At 0.025 mM [K⁺], RPU values was found to be 0.13. There was about 3.8 fold change in RPU from 0 mM to 0.4 mM [K⁺]. As expected, kdpFp is turned off at high [K⁺] due to inhibition of kinase activity of kdpD by K⁺.

Relative fluorescence measurement

Figure 5 & 6. Activity of kdpFp in E. coli DH10B in different K⁺ concentrations. 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. Cells were pre-cultured, washed and sub-cultured as previously described in RPU measurement. Measurement took place when OD₆₀₀=0.4. 2 other measurements were taken every 15 mins afterwards for GFP synthesis rate. Error bar are represented as SEM.

Both C and G mutants similarly expressed higher fluorescence compared to the wild type and the A mutant. As the [K⁺] went up, the activity of kdpFp decreased. 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. Strength of both C and G mutants, at 0 mM [K⁺], is about 1.7 times stronger than A mutant and wild type promoter. At 0.2 mM [K⁺], both C and G mutants are still 1.7 times strong that wild type promoter, but they are 3 times stronger when compared to A mutant. This might be caused by the difference in binding affinity between RNA polymerase and kdpFp variants (Brewster, 2012). We also found that the dynamic range of our promoters is between 0 to 0.1 mM of K⁺.

Figure 8. Comparison between the activities of kdpFp[-15, T>G] in DH10B and TK2240 strain. Cells were pre-cultured, washed and sub-cultured as previously described in RPU measurement. Measurement took place when OD₆₀₀=0.4. Error bars are represented as SEM.

At [K⁺] lower than 0.0125 mM, the acitivity of G mutant in DH10B was significantly greater than the one in TK2240 strain. Activity of kdpFp in DH10B strain decreased over the concentration, while the one in TK2240 remained stable. After passing the [K⁺] at 0.05 mM, the activity of kdpFp in TK2240 strain exceeded the one in the DH10B strain. Since TK2240 is defective in Trk and Kup system, it can only rely on Kdp system for K⁺ uptake, so an higher promoter activity after 0.05 mM [K⁺] is observed. While at the [K⁺] lower than 0.0125 mM, the distinct activity might be due to the greater need of kdpFABC complex for K⁺ scavenge, which deplete the energy of TK2240 in making other protein.

Considerations for replicating the experiments

a. OD₆₀₀= 0.4 referring to mid log phase of E. coli in K minimal medium
b. Dilution has always been done to make the OD₆₀₀ values of start culture around the same before subculturing in different concentration of K⁺.

Future Plan

In the interest of providing an efficient and accessible device that can identify the concentration of K+ ion into real field, we had a future plan on optimizing the device prepared using a paper-based cell-free transcription-translation (TX-TL) system for our construct.

References

Brewster, R. C., Jones, D. L., & Phillips, R. (2012). Tuning promoter strength through RNA polymerase binding site design in Escherichia coli.

Epstein, W., & Kim, B. S. (1971). Potassium transport loci in Escherichia coli K-12. Journal of Bacteriology, 108(2), 639-644.

Jung, K., Tjaden, B., & Altendorf, K. (1997). Purification, reconstitution, and characterization of KdpD, the turgor sensor of Escherichia coli. Journal of Biological Chemistry, 272(16), 10847-10852.

Jung, K., Veen, M., & Altendorf, K. (2000). K+ and ionic strength directly influence the autophosphorylation activity of the putative turgor sensor KdpD of Escherichia coli. Journal of Biological Chemistry, 275 (51), 40142-40147.

Jung, K., Krabusch, M., & Altendorf, K. (2001). Cs+ Induces the kdp operon of Escherichia coli by Lowering the Intracellular K+ Concentration. Journal of bacteriology, 183(12), 3800-3803.

Laermann, V., Ćudić, E., Kipschull, K., Zimmann, P., & Altendorf, K. (2013). The sensor kinase KdpD of Escherichia coli senses external K+. Molecular microbiology, 88(6), 1194-1204.

Laimins, L. A., Rhoads, D. B., & Epstein, W. (1981). Osmotic control of kdp operon expression in Escherichia coli. Proceedings of the National Academy of Sciences, 78 (1), 464-468.

Narayanan, A., Paul, L. N., Tomar, S., Patil, D. N., Kumar, P., & Yernool, D. A. (2012). Structure-function studies of DNA binding domain of response regulator KdpE reveals equal affinity interactions at DNA half-sites. PloS one, 7(1), e30102.

Polarek, J. W., Williams, G., & Epstein, W. (1992). The products of the kdpDE operon are required for expression of the Kdp ATPase of Escherichia coli. Journal of bacteriology, 174 (7), 2145-2151.

Roe, A. J., McLaggan, D., O’Byrne, C. P., & Booth, I. R. (2000). Rapid inactivation of the Escherichia coli Kdp K+ uptake system by high potassium concentrations. Molecular microbiology, 35(5), 1235-1243.

Sugiura, A., Nakashima, K., Tanaka, K., & Mizuno, T. (1992). Clarification of the structural and functional features of the osmoregulated kdp operon of Escherichia coli. Molecular microbiology, 6(13), 1769-1776.

Voelkner, P., Puppe, W., & Altendorf, K. (1993). Characterization of the KdpD protein, the sensor kinase of the K+‐translocating Kdp system of Escherichia coli. European Journal of Biochemistry, 217(3), 1019-1026.

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.

Yan, H., Fukamachi, T., Saito, H., & Kobayashi, H. (2011). Expression and activity of Kdp under acidic conditions in Escherichia coli. Biological and Pharmaceutical Bulletin, 34(3), 426-429.

Zhang, L., Jiang, W., Nan, J., Almqvist, J., & Huang, Y. (2014). The Escherichia coli CysZ is a pH dependent sulfate transporter that can be inhibited by sulfite. Biochimica et Biophysica Acta (BBA)-Biomembranes, 1838(7), 1809-1816.