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<div id= "page_title"><h1>Potassium Sensor</h1>
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<div id= "page_title"><h1>Potassium Sensor - <i>P<sub>kdpF</sub></i></h1></div>
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                <a href="https://2015.igem.org/Team:HKUST-Rice/Modeling"><img src="https://static.igem.org/mediawiki/2015/7/7a/HKUST-Rice15_rightarrow.png">
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<p style="color:#5570b0; font-size: 130%"> Potassium sensor - Modeling </p></a>
 
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<h1>Potassium as a Macro-nutrient</h1>
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<h1><i>E. coli</i> that glows in paucity of K<sup>+</sup> - at a glance</h1>
<p>Potassium is an essential plant macronutrients as it has numerous roles in plants and it is required for plant growth and development. Some of its important roles include the regulation of opening and closing of stomata which therefore regulates water (osmoregulation) and CO<sub>2</sub>. It is also essential in starch synthesis and protein synthesis. Moreover, it activates many growth related enzymes in plants. Hence, the deficiency of K+ ion will result in abnormalities in plant growth and metabolism.</p>
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<h1>What we try to achieve</h1>
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<p>On account of crucial impact that K<sup>+</sup> can contribute to the plant performance, it is fundamental to determine its concentration in soil in order to provide the proper amount of additional potassium that must be added to the plant by any particular fertilizers.  
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Our aim is to engineer a Potassium sensor that can detect a range of K<sup>+</sup> concentration in the soil to ensure the suitable soil condition for the plant fitness. </p>
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<p style="font-size:110%; padding-left:2%; padding-right: 2% ; height'90px';"><strong>A.</strong> <i>E. coli</i> engineered with <a href="http://parts.igem.org/Part:BBa_K1682009"target="_blank">BBa_K1682009</a> functions as a potassium biosensor. High concentrations of K<sup>+</sup> indirectly represses the promoter <i>K<sub>kdpF</sub></i> and decreases the expression of GFP.</p>
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<p style="font-size:110%; padding-left:2;height:'90px'; padding-right: 2%"  ><strong>B.</strong> The potassium sensing promoter <a href="http://parts.igem.org/Part:BBa_K1682004"target="_blank">BBa_K1682004</a> 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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<p><ul style="text-align:left; font-size:1.5em; line-height= 1.5em; font-family: 'Helvetica Neue', Helvetica, sans-serif;"><li>K<sup>+</sup> is an essential plant macronutrient and plays vital role for maintaining high crop yield.</li>
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<li>Our biosensor <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1682009">BBa_K1682009</a> monitors K<sup>+</sup> concentration.</li>
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<li>Activity of K<sup>+</sup> sensing promoter (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1682004">BBa_K1682004</a>) was measured in Relative Promoter Unit. It can be reliably reused.</ul></p>
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<h1>Potassium sensor Design</h1>
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<h1><i>P<sub>kdpF</sub></i> and our engineered K<sup>+</sup> sensor BBa_K1682009 - the full story</h1>
<p>We utilized <i>kdpFp</i>, a promoter located upstream of <i>kdpFABC</i> operon in <i>Escherichia coli</i> (<i>E. coli</i>) which works under low K<sup>+</sup> concentration in pursuance of a precise and functional Potassium Sensor. However, there is an illegal EcoRI site in <i>KdpFp</i> promoter, thus, we attempted to remove that illegal site by constructing G-Mutant <i>kdpFp</i>, C-Mutant <i>kdpFp</i> and A-Mutant <i>kdpFp</i>. Those 3 promoters have the illegal site removed by G (guanine), C (Cytosine) and A (Adenine).
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<p>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<sup>+</sup> ion will result in abnormalities in plant growth and metabolism. Our aim is to engineer a potassium sensor in <i>Escherichia coli</i> and detect the lack of K<sup>+</sup> in soil. To this end, we engineered <i>P<sub>kdpF</sub></i>, a promoter activated under low [K<sup>+</sup>] condition, and fused it with <i>gfp</i> (<i>gfpmut3b</i>).</p>
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<p class="subTitle">Endogenous potassium sensing system in <i>E. coli</i></p>
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<h1>Measurement and Characterization</h1>
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<p style="font-size:110%; padding-left:6%;"><strong>Figure 1. The Kdp K<sup>+</sup> uptake system in  <i>E. coli</i>.</strong></p>
<p>Upon different concentration of K<sup>+</sup>, Potassium sensor will show different fluorescence level due to distinctive effect of K<sup>+</sup> ion to <i>kdpFp</i>.
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We characterized kdpFp by using RPU as standard unit. The measurement results were obtained with FACS (Fluorescence-activated cell sorting) and EnVision multilabel reader.</p>
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<h1>Achievement</h1>
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<p>Hitherto, our team has finished characterizing all the constructs and contemplated the activity of the promoters over a varying range of K<sup>+</sup> concentration.</p>
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<h1>Mechanism</h1>
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<p>In our project, we use the native potassium ion transport system in <i>Escherichia coli</i> (<i>E. coli</i>), Kdp system as our potassium sensing part. The Kdp system is composed of two major parts, KdpFABC, a high-affinity potassium transporter as well as two types of regulatory proteins, a sensory kinase KdpD and a response regulator KdpE. KdpD and KdpE works together as a two-component system, tracking and responding to the intra and extracellular potassium level then interacting with the KdpFABC encoding operon. <i>kdpFABC</i> operon is up-regulated under low potassium ion concentration and is inhibited under high concentration. <br><br>
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KdpD, which is a trans-membrane protein, auto-phosphorylates itself, also phosphorylates and dephosphorylates KdpE. Low concentration of potassium ions favors the phosphorylation of KdpE, which then gives rise to the enhancement of the level of phosphorylated KdpE, and as a result, triggers the up-regulation of <i>kdpFABC</i> operon.<br><br>
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As our potassium-sensing device, we adopt the promoter <i>kdpFp</i> from <i>kdpFABC</i> operon. The sequence was obtained by oligos, we then combine the promoter with the downstream GPF generator using biobrick RFC 10 so that the change of the promoter activity in different potassium level can be detected and characterized.
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<h1>Limitations</h1>
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<p>There are two major limitations in making use of the Kdp system as our potassium-sensing module. The first main concern is that the promoter <i>kdpFp</i> contains the EcoRI illegal site. While the second concern is about the background noise contributed by other native constitutive potassium transport systems of <i>E. coli</i>, including trk and Kup systems, which are potassium ions influx systems and are expected to lower the activity of our promoter <i>kdpFp</i>.  
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<h1>Solutions to the limitations</h1>
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<p>We have come up with solutions to tackle the aforementioned limitations of Kdp system. For the EcoRI illegal site inside the promoter, we ordered 4 different versions of <i>kdpFp</i>, one of them is the wild-type promoter; for the other three, they have one base-pair at -15 site, where the illegal site locate, changes from thymine (T) to cytosine (C), guanine (G) and adenine (A) respectively. This make the three promoters into three different mutants, we denote them as A-mutant, G-mutant and C-mutant respectively. All the mutants, thereby, have their illegal site removed.
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<h1>Result obtained</h1>
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<p>Lorem ipsum dolor sit amet, pro aeque temporibus eu, eum qualisque assueverit te. Ad est admodum epicuri suscipit, te alterum aliquando adversarium usu, pro ex omnesque luptatum comprehensam. In vix alia percipit gloriatur, no ferri lorem aliquando cum. Fugit concludaturque sed ne, ea sumo dico adolescens eos, quo eu pertinax expetendis. An his omnes instructior, vide possim eam id. Te cum enim sale offendit, vocent copiosae luptatum ut per.
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Eam in alienum accusamus, et probo reque vix. Vivendum necessitatibus qui ad, no vis enim veniam perpetua. Eu pri habemus senserit, dicit tation expetenda usu et. Sea eu dolor deserunt dissentias, sed an oportere moderatius assueverit. Usu te tation gloriatur, vidit tollit utinam mea id.</p>
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<p>Lorem ipsum dolor sit amet, pro aeque temporibus eu, eum qualisque assueverit te. Ad est admodum epicuri suscipit, te alterum aliquando adversarium usu, pro ex omnesque luptatum comprehensam. In vix alia percipit gloriatur, no ferri lorem aliquando cum. Fugit concludaturque sed ne, ea sumo dico adolescens eos, quo eu pertinax expetendis. An his omnes instructior, vide possim eam id. Te cum enim sale offendit, vocent copiosae luptatum ut per.
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, et probo reque vix. Vivendum necessitatibus qui ad, no vis enim veniam perpetua. Eu pri habemus senserit, dicit tation expetenda usu et. Sea eu dolor deserunt dissentias, sed an oportere moderatius assueverit. Usu te tation gloriatur, vidit tollit utinam mea id.</p>
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<p><i>E. coli</i> has multiple native K<sup>+</sup> 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<sup>+</sup> concentration, both extracelluarly and intracellularly, KdpD transphosphorylates KdpE using its own phosphate. The phospho-KdpE is then capable of activating expression of the <i>kdpFABC</i> operon, which codes for a transporter complex that is activated by low K<sup>+</sup> concentration. Apart from that, <i>E. coli</i>  also has constitutively expressed Trk and Kup transporters for K<sup>+</sup> uptake.
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<p style = "font-size:110%">*The above text is our summarized understanding on K<sup>+</sup>-sensing system using information from EcoCyc. (Keseler et al., 2013). Please refer to our references section below for a full list of references cited.</p>
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<p  class="subTitle">Design and Testing of potassium sensing Device</p>
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<p style="font-size:110%; padding-left:6%;"><strong>Figure 2. Construction and Testing of <i>P<sub>kdpF</sub></i>.</strong> A) Positions of base substitutions to standardize <i>P<sub>kdpF</sub></i> into RFC10 format. B) Single time point transfer curve for <i>P<sub>kdpF</sub></i> variants along a gradient of [K<sup>+</sup>]. C) Relative GFP synthesis rate calculated from 3 measurement time points. Error bar present SEM from 3 biological replicates.</p></div>
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<p>To construct a potassium-sensing device, we cloned the promoter upstream of <i>kdpFABC</i> operon, <i>P<sub>kdpF</sub></i></a>, and fused it with a translation unit for GFP reporter <a href="http://parts.igem.org/Part:BBa_E0240"target="_blank">BBa_E0240</a> in BioBrick RFC10 standard. The promoter activity can then be reported by the GFP level under different K<sup>+</sup> concentrations.</p>
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<p>However, <i>P<sub>kdpF</sub></i> contains an illegal <i>EcoR</i>I site that prohibits standard assembly. We tackled this by constructing and testing 3 mutated versions with A (<a href ="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1682002"target="_blank">BBa_K1682002</a>), C (<a href ="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1682003"target="_blank">BBa_K1682003</a>), or G (<a href ="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1682004"target="_blank">BBa_K1682004</a>) substituting the orignal T. Our results showed that all 3 variants are functional and sense [K<sup>+</sup>] from 0 to 0.1 mM. The C and G mutants had higher maximum promoter activities than the WT or A mutant. <i>P<sub>kdpF</sub></i>[-15,T>G] performed well in both strength and reliablity and was therefore used in subsequent experiments.</p>
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<p  class="subTitle">Relative Promoter Unit Measurement of <i>P<sub>kdpF</sub></i>[-15,T>G]</p>
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<p style="font-size:110%; padding-left:6%;"><strong>Figure 3. Relative promoter units (RPU) of <i>P<sub>kdpF</sub></i>[-15,T>G] under different [K<sup>+</sup>].</strong> Error bar present SEM from 3 independent experiments on different days.</p></div>
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<p>We decided to report the promoter activites of <i>P<sub>kdpF</sub></i>[-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 <i>P<sub>kdpF</sub></i>[-15,T>G] in different [K<sup>+</sup>] were measured and compared to that by <a href="http://parts.igem.org/Part:BBa_I20260"target="_blank">BBa_I20260</a> following a modified protocol from Kelly et. al (2009) (see below), and was found to be ~0.5 RPU at 0 mM K<sup>+</sup> and ~0.13 RPU 0.025 mM K<sup>+</sup>. From 0 - 0.4 mM K<sup>+</sup>, there is a 3.8 fold change in RPU.</p>
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<p  class="subTitle">Interference from other Endogenous Systems</p>
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<p style="font-size:110%; padding-left:6%;"><strong>Figure 4. Comparison between the activities of <i>P<sub>kdpF</sub></i>[-15, T>G] in DH10B and TK2240 strain. </strong> Error bar present SEM from 3 biological replicates.</p>
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<p>The low-affinity K<sup>+</sup> transport systems Trk and Kup native to <i>E. coli</i> are constitutively expressed (Epstein & Kim, 1971). Laermann et al. (2013) discovered that knocking out the two systems in the strain TK2240 <i>(kdp+ Δtrk Δkup)</i> will result in a increase in expression of the <i>kdpFABC</i> system. We repeated that comparison using DH10B with our <i>P<sub>kdpF</sub></i>[-15,T>G] promoter but obtained different results - below 0.0125 mM K<sup>+</sup>, the activity of the promoter in DH10B was significantly greater than that in TK2240. Only when [K<sup>+</sup>] > 0.05mM would we be able to observe stronger promoter activities in TK2240. We are uncertain about what causes the discrepancies in the comparisons.</p>
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<h1>Future Plan</h1>
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<p>In the interest of providing an efficient and accessible device that can identify the [K<sup>+</sup>] into real field, we plan to optimize our construct in a device using a paper-based cell-free transcription-translation (TX-TL) system.</p>
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<h2>Materials and Methods</h2>
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<p>Please refer to <a href ="https://2015.igem.org/Team:HKUST-Rice/Protocol">our protocol page for the materials and methods used in characterization.</a></p>
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<h2>References</h2>
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<p style="font-size:125%">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. <i>Journal of biological engineering</i>, 3(1), 4.
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<br><br>International Plant Nutrition Institute. (1998). Functions of Potassium in Plants. <i>Better Crops</i>, 82(3).
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<br><br>Keseler et al. (2013), EcoCyc: fusing model organism databases with systems biology, <i>Nucleic Acids Research</i> 41: D605-12.</p>
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<h2>References on potassium uptake and regulation systesm in <i>E. coli</i></h2>
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<p style="font-size:125%">Epstein, W., & Kim, B. S. (1971). Potassium transport loci in <i>Escherichia coli</i> K-12. <i>Journal of Bacteriology, 108</i>(2), 639-644.
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<br><br>Jung, K., Tjaden, B., & Altendorf, K. (1997). Purification, reconstitution, and characterization of KdpD, the turgor sensor of <i>Escherichia coli.</i> <i>Journal of Biological Chemistry, 272</i>(16), 10847-10852.
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<br><br>Jung, K., Veen, M., & Altendorf, K. (2000). K<sup>+</sup> and ionic strength directly influence the autophosphorylation activity of the putative turgor sensor KdpD of <i>Escherichia coli</i>. <i>Journal of Biological Chemistry, 275 </i>(51), 40142-40147.
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<br><br>Jung, K., Krabusch, M., & Altendorf, K. (2001). Cs+ Induces the kdp operon of <i>Escherichia coli</i> by Lowering the Intracellular K<sup>+</sup> Concentration. <i>Journal of bacteriology, 183</i>(12), 3800-3803.
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<br><br>Laermann, V., Ćudić, E., Kipschull, K., Zimmann, P., & Altendorf, K. (2013). The sensor kinase KdpD of <i>Escherichia coli</i> senses external K<sup>+</sup>. <i>Molecular microbiology, 88</i>(6), 1194-1204.
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<br><br>Laimins, L. A., Rhoads, D. B., & Epstein, W. (1981). Osmotic control of kdp operon expression in <i>Escherichia coli. Proceedings of the National Academy of Sciences, 78 </i>(1), 464-468.
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<br><br>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. <i>PloS one, 7</i>(1), e30102.
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<br><br>Polarek, J. W., Williams, G., & Epstein, W. (1992). The products of the kdpDE operon are required for expression of the Kdp ATPase of <i>Escherichia coli. Journal of bacteriology, 174 </i>(7), 2145-2151.
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<br><br>Roe, A. J., McLaggan, D., O’Byrne, C. P., & Booth, I. R. (2000). Rapid inactivation of the <i>Escherichia coli</i> Kdp K<sup>+</sup> uptake system by high potassium concentrations. <i>Molecular microbiology, 35</i>(5), 1235-1243.
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<br><br>Sugiura, A., Nakashima, K., Tanaka, K., & Mizuno, T. (1992). Clarification of the structural and functional features of the osmoregulated kdp operon of <i>Escherichia coli. Molecular microbiology, 6</i>(13), 1769-1776.
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<br><br>Voelkner, P., Puppe, W., & Altendorf, K. (1993). Characterization of the KdpD protein, the sensor kinase of the K<sup>+</sup>‐translocating Kdp system of <i>Escherichia coli. European Journal of Biochemistry, 217</i>(3), 1019-1026.
 +
<br><br>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. <i>Journal of bacteriology, 174</i>(7), 2152-2159.
 +
<br><br>Yan, H., Fukamachi, T., Saito, H., & Kobayashi, H. (2011). Expression and activity of Kdp under acidic conditions in <i>Escherichia coli. Biological and Pharmaceutical Bulletin, 34</i>(3), 426-429.
 +
<br><br>Zhang, L., Jiang, W., Nan, J., Almqvist, J., & Huang, Y. (2014). The <i>Escherichia coli</i> CysZ is a pH dependent sulfate transporter that can be inhibited by sulfite. <i>Biochimica et Biophysica Acta (BBA)-Biomembranes, 1838</i>(7), 1809-1816. </p>
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{{HKUST-Rice Directory}}

Latest revision as of 02:27, 19 September 2015



Potassium Sensor - PkdpF

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


A. E. coli engineered with BBa_K1682009 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_K1682009 monitors K+ concentration.
  • Activity of K+ sensing promoter (BBa_K1682004) was measured in Relative Promoter Unit. It can be reliably reused.


PkdpF and our engineered K+ sensor BBa_K1682009 - 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 the 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

image caption

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 phospho-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

image caption

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]

image caption

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 from Kelly et. al (2009) (see below), and was found to be ~0.5 RPU at 0 mM 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

image caption

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 comparison 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.


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.