E. coli that glows in paucity of K⁺ - at a glance

A. E. coli engineered with BBa_K1682005 functions as a potassium biosensor. High concentrations of K+ indirectly represses the promoter KkdpF and decreases the expression of GFP.		B. The potassium sensing promoter BBa_K1682004 can detect a gradient of K+ concentrations and its activity can be reported in Relative Promoter Units (RPU).

• K⁺ is an essential plant macronutrient and plays vital role for maintaining high crop yield.
• Our biosensor BBa_K1682005 monitors K⁺ concentration.
• Activity of K⁺ sensing promoter (BBa_K1682000) was measured in Relative Promoter Unit. It can be reliably reused.

P_kdpF and our engineered K⁺ sensor BBa_K1682005 - the full story

Potassium is an essential plant macronutrient as it is required for photosynthesis, osmoregulation, stomatal control, sugar and protein synthesis. (CITATION) The deficiency of K⁺ ion will result in abnormalities in plant growth and metabolism. Our aim is to engineer a potassium sensor in Escherichia coli and detect lack of K⁺ in soil. To this end, we engineered P_kdpF, a promoter activated under low [K⁺] condition, and fused it with gfp (gfpmut3b).

Endogenous K⁺ sensing system in E. coli

Figure 2. The kdp K⁺ uptake system in E.coli.

E. coli has multiple native K⁺ sensing and uptake systems that we could put to use. Among them, we chose the KdpDE two-component system (TCS). It contains a membrane-bound kinase KdpD and a cytoplasmic response regulator KdpE. Stimulated by low K⁺ concentration, both extracelluarly and intracellularly, KdpD transphosphorylates KdpE using its own phosphate. The phosph-KdpE is then capable of activating expression of the kdpFABC operon, which codes for a transporter complex that is activated by low K⁺ concentration.

Apart from that, E. coli also has constitutively expressed Trk and Kup transporters for K⁺ uptake.

*The above text is our summarized understanding on K⁺-sensing system using information from EcoCyc. (Keseler et al., 2013). Please refer to our references section below for a full list of references cited.

Design of K⁺-sensing Device

To make a potassium-sensing device, we obtained the promoter upstream of kdpFABC operon, P_kdpF, and fused it with a translation unit for GFP reporter BBa_E0240 in BioBrick RFC10 standard. The promoter activity was reported by the GFP level under different K⁺ concentrations.

EcoRI Illegal Site

To make P_kdpF 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 P_kdpF 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 P_kdpF may not be truly reflected due to its masking by the Trk and Kup systems. We decided to measure the activity of P_kdpF 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 P_kdpF 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

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, P_kdpF is turned off at high [K⁺] due to inhibition of KdpD kinase activity by K⁺.

Relative fluorescence measurement

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 P_kdpF 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 P_kdpF variants (Brewster, 2012). The dynamic range of our promoters is between 0 to 0.1 mM of K⁺.

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 P_kdpF in DH10B strain decreased with increasing concentrations, while it remained stable in TK2240. Above 0.05 mM [K⁺], the activity of P_kdpF 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.

Keseler et al. (2013), EcoCyc: fusing model organism databases with systems biology, Nucleic Acids Research 41: D605-12.

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.