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 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.

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 and Testing of K⁺-sensing Device

To construct a potassium-sensing device, we cloned 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 can then be reported by the GFP level under different K⁺ concentrations.

However,P_kdpF 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. They should have different strengths due to different binding energies between the promoter and RNA polymerase (Brewster, 2012).

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, which did not fully agree with predicted RNAP binding affinites. P_kdpF[-15,T>G] performed well in both strength and reliablity and was therefore used in subsequent experiments.

Relative Promoter Unit Measurement of P_kdpF[-15,T>G]

We decided to report the promoter activites of P_kdpF[-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 P_kdpF[-15,T>G] in different [K⁺] were measured and compared to that by BBa_I20260 following a modified protocol from Kelly et. al (CITATION), 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.

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

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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.

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