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<p style="font-size:110%; padding-left:2; padding-right: 2%"><strong>B.</strong> The potassium sensing promoter BBa_K1682004 can detect a gradient of K<sup>+</sup> concentrations</strong> and its activity can be reported in Relative Promoter Units (RPU).</p>
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<p style="font-size:110%; padding-left:2; padding-right: 2%"><strong>B.</strong> The potassium sensing promoter BBa_K1682004 can detect a gradient of K<sup>+</sup> concentrations</strong> and its activities were reported in Relative Promoter Units (RPU).</p>
 
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Revision as of 06:27, 18 September 2015



Potassium Sensor - PkdpF

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.


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. (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 PkdpF, a promoter activated under low [K+] condition, and fused it with gfp (gfpmut3b).


Endogenous K+ sensing system in E. coli

image caption

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

image caption image caption

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. 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. 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]

image caption

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 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.


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

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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+.

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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.

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.