Team:HKUST-Rice/Potassium Sensor
Potassium Sensor - PkdpF
Potassium as a Macro-nutrient
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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 using Escherichia coli as chassis that can detect a range of [K+] in the soil to monitor soil condition for the plant. We utilized PkdpF, a promoter activated under low [K+] condition, and gfpmut3b as reporter signal for the sensor. |
Figure 1. Engineered E. coli as potassium biosensor. |
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+] conditions (Laimins et al., 1981).
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 K+ (Jung, 2000; Jung, 2001; Roe, 2000; Yan, 2011a; Laermann, 2013). KdpD phosphorylates KdpE upon low [K+] (Voelkner, 1993; Puppe, 1996; Jung, 1997a; Jung, 2000). Under a decrease in [K+], KdpD kinase activity is enhanced, causing an increase in phospho-KdpE. Phosphorylated KdpE turns on the expression of kdpFABC (Zhang, 2014a; Laermann, 2013).
Figure 2. The kdp K+ uptake system in E.coli.
Design of K+ Sensing Device
To make a potassium-sensing device, we obtained the promoter upstream of kdpFABC operon, PkdpF and combined it with a GFP reporter BBa_E0240 in BioBrick RFC10 standard. The promoter activity was reported by the GFP level under different [K+].
Figure 3. K+ sensing construct with reporter, BBa_K1682005.
EcoRI Illegal Site
To make PkdpF compatible with RFC10 standard and, as so, readily accessible to iGEM community, we designed variants of it with the EcoRI site removed. We mutated the thymine at -15 position to adenine, cytosine and guanine. 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 comprehensive knowledge in the activity and working range of the four promoters. For convenience, we denote them as A mutant (BBa_K1682002), C mutant (BBa_K1682003) and G mutant (BBa_K1682004) respectively in the following context.
Figure 4. Comparison of relative RNAP binding affinity at mutated site of wild type PkdpF and its variants.
Interference from other Endogenous Systems
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). Laermann et al. (2013) found that the expression level of kdpFABC is generally higher in a strain without the Trk and Kup systems. In other words, the activity of PkdpF may not be truly reflected due to its masking by the Trk and Kup systems. We decided to measure the activity of PkdpF in E. coli TK2240 (kdp+ Δtrk Δkup) strain, so that we are able to characterize it in a more accurate manner.
Measurement and Characterization
To make it more easily used by others, we characterized PkdpF by relative promoter unit (RPU) measurement. Additionally, relative fluorescence measurement has also been done to compare the activities between wild type promoters and 3 variants.
Relative promoter unit measurement
Figure 5. Relative promoter unit (RPU) of PkdpF[-15,T>G] at different [K+]. Cells were pre-cultured in K115 medium overnight at 37°C. The cells were washed 3 times in 0.8% 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 presented in SEM.
At the lowest [K+], the strength of the G mutant promoter was found to be approximately 0.5 RPU. RPU of the promoter decreases as [K+] increases. 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, PkdpF is turned off at high [K+] due to inhibition of KdpD kinase activity by K+.
Relative fluorescence measurement
Figure 6 & 7. Activity of PkdpF in E. coli DH10B in different [K+].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 presented in SEM.
Both C and G mutants expressed higher fluorescence compared to the wild type and the A mutant. As the [K+] went up, the activity of PkdpF decreased. The expression levels of both the C and G mutant promoters were significantly higher than the wild type promoter, while the A mutant was always the lowest. At 0 mM [K+], both the C and G mutants expressed fluorescence which was about 1.7 times higher than the A mutant and wild type promoter. At 0.2 mM [K+], expression of both C and G mutants were 1.7 times higher than the wild type promoter, and 3 times higher compared to the A mutant. This might be caused by the difference in binding affinity between RNA polymerase and PkdpF variants (Brewster, 2012). The dynamic range of our promoters is between 0 to 0.1 mM of K+.
Figure 8. Comparison between the activities of PkdpF[-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.
As expected, the promoter activity is shown to be different in the absence of Trk and Kup system. At [K+] below 0.0125 mM, the acitivity of G mutant in DH10B was significantly greater than that in TK2240 strain. Activity of PkdpF in DH10B strain decreased with increasing concentrations, while it remained stable in TK2240. Above 0.05 mM [K+], the activity of PkdpF in TK2240 strain exceeded that in the DH10B strain. Since TK2240 is defective in its Trk and Kup systems, it can only rely on the Kdp system for K+ uptake, thereby explaining the higher promoter activity observed beyond 0.05 mM [K+].
Future Plan
In the interest of providing an efficient and accessible device that can identify the [K+] into real field, we plan to optimize our construct in a device using a paper-based cell-free transcription-translation (TX-TL) system.
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.
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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.