Team:HKUST-Rice/Potassium Sensor/dummy1
Potassium Sensor - PkdpF
E. coli that glows in paucity of K+ - at a glance
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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 activities were 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.
PkdpF 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. (IPNI, 1998) 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 PkdpF, a promoter activated under low [K+] condition, and fused it with gfp (gfpmut3b).
Endogenous potassium sensing system in E. coli
Figure 1. 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 and Testing of potassium sensing Device
Figure 2. Construction and Testing of PkdpF. A) Positions of base substitutions to standardize PkdpF into RFC10 format. B) Single time point transfer curve for PkdpF variants along a gradient of [K+]. C) Relative GFP synthesis rate calculated from 3 measurement time points. Error bar present SEM from 3 biological replicates.
To construct a potassium-sensing device, we cloned the promoter upstream of kdpFABC operon, PkdpF, and fused it with a translation unit for GFP reporter BBa_E0240 in BioBrick RFC10 standard. The promoter activity can then be reported by the GFP level under different K+ concentrations.
However,PkdpF contains an illegal EcoRI site that prohibits standard assembly. We tackled this by constructing and testing 3 mutated versions with A (BBa_K1682002) , C (BBa_K1682003), or G (BBa_K1682004) substituting the orignal T. Our results showed that all 3 variants are functional and sense [K+] from 0 to 0.1 mM. The C and G mutants had higher maximum promoter activities than the WT or A mutant. PkdpF[-15,T>G] performed well in both strength and reliablity and was therefore used in subsequent experiments.
Relative Promoter Unit Measurement of PkdpF[-15,T>G]
Figure 3. Relative promoter units (RPU) of PkdpF[-15,T>G] under different [K+]. Error bar present SEM from 3 independent experiments on different days.
We decided to report the promoter activites of PkdpF[-15,T>G] in Relative Promoter Units (RPU) because this will allow future users to compare its promoter strength with that from another promoter, and perhaps, further improve this part. The activities of PkdpF[-15,T>G] in different [K+] were measured and compared to that by BBa_I20260 following a modified protocol (see below) from Kelly et. al (2009), and was found to be ~0.5 RPU at 0mM K+ and ~0.13 RPU 0.025 mM K+. From 0 - 0.4 mM K+, there is a 3.8 fold change in RPU.
Interference from other Endogenous Systems
Figure 4. Comparison between the activities of PkdpF[-15, T>G] in DH10B and TK2240 strain. Error bar present SEM from 3 biological replicates.
The low-affinity K+ transport systems Trk and Kup native to E. coli are constitutively expressed (Epstein & Kim, 1971). Laermann et al. (2013) discovered that knocking out the two systems in the strain TK2240 (kdp+ Δtrk Δkup) will result in a increase in expression of the kdpFABC system. We repeated that comparsion using DH10B with our PkdpF[-15,T>G] promoter but obtained different results - below 0.0125 mM K+, the activity of the promoter in DH10B was significantly greater than that in TK2240. Only when [K+] > 0.05mM would we be able to observe stronger promoter activities in TK2240. We are uncertain about what causes the discrepancies in the comparisons.
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.
Materials and Methods
Please refer to our protocol page for the materials and methods used in characterization.
References
Kelly, J. R., Rubin, A. J., Davis, J. H., Ajo-Franklin, C. M., Cumbers, J., Czar, M. J., ... & Endy, D. (2009). Measuring the activity of BioBrick promoters using an in vivo reference standard. Journal of biological engineering, 3(1), 4.
International Plant Nutrition Institute. (1998). Functions of Potassium in Plants. Better Crops, 82(3).
Keseler et al. (2013), EcoCyc: fusing model organism databases with systems biology, Nucleic Acids Research 41: D605-12.
References on potassium uptake and regulation systesm in E. 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.