Difference between revisions of "Team:HKUST-Rice/Potassium Sensor"

 
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<div id= "page_title"><h1>Potassium Sensor - <i>P<sub>kdpF</sub></i></h1></div>
 
<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">
 
                 <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>
 
<p style="color:#5570b0; font-size: 130%"> Potassium sensor - Modeling </p></a>
 
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<h1><i>E. coli</i> that glows in paucity of K<sup>+</sup> - at a glance</h1>
<h1>Potassium as a Macro-nutrient</h1>
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<img  src="https://static.igem.org/mediawiki/2015/4/42/Team_HKUST-Rice_2015_potassium_figure_1.png "style="width:100%;">
<p style="font-size:110%; padding-left:6%;"><strong>Figure 1. Engineered <i>E. coli</i> as potassium biosensor.</strong></p></div>
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<p>Potassium is an essential plant macronutrient as it has numerous important roles in plants including osmoregulation,
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CO<sub>2</sub> regulation, starch and protein synthesis. The deficiency of K<sup>+</sup> ion will result
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in abnormalities in plant growth and metabolism. Our aim is to engineer a potassium sensor using <i>Escherichia coli</i> as chassis that can detect a range of [K<sup>+</sup>] in the soil to monitor soil condition for the plant. We utilized <i>P<sub>kdpF</sub></i>, a promoter activated at under low [K<sup>+</sup>] condition, and GFPmut3b as reporter signal for the sensor.</p>
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<img id="Kgraph" src="https://static.igem.org/mediawiki/2015/9/90/HKUST-Rice15_%28log_10%29_RPU_of_kdpFp--15%2CT_G-_in_DH10B_-RPU-.png" style="width:100%">
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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>Endogenous K<sup>+</sup> sensing system in <i>E. coli</i></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>The potassium ion uptake in <i>E. coli</i> is regulated by several systems under different conditions.
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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>
The potassium ion transporters, Trk and Kup are constitutively expressed (Epstein & Kim, 1971) while KdpFABC, another
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transporter is activated under low [K<sup>+</sup>] conditions (Laimins et al., 1981).</p>
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<p>The <i>kdpFABC</i> operon is controlled by
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the KdpDE two-component system (TCS) which consists of KdpD, a membrane-bound
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sensor kinase, and KdpE, a cytoplasmic response regulator (Polarek, 1992; Walderhaug, 1992). KdpD is stimulated by both intracellular and extracellular
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K<sup>+</sup> (Jung, 2000; Jung, 2001; Roe, 2000; Yan, 2011a; Laermann, 2013). KdpD phosphorylates KdpE upon low [K<sup>+</sup>] (Voelkner, 1993; Puppe, 1996; Jung, 1997a; Jung, 2000).
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Under an increase in [K<sup>+</sup>], KdpD phosphatase activity will be enhanced, causing a decrease in phospho-KdpE
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and <i>kdpFABC</i> expression. Phosphorylated KdpE turns on the expression of <i>kdpFABC</i> (Zhang, 2014a; Laermann, 2013).</p>
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<div class="des">
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<p style="font-size:110%; padding-left:6%;"><strong>Figure 2. The kdp K<sup>+</sup> uptake system in <i>E.coli</i> and the potassium biosensor design.</strong></p></div>
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<h1>Design of K<sup>+</sup> sensing Device</h1>
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<p>To make a potassium-sensing device, we obtained the promoter upstream of <i>kdpFABC</i> operon, <a href ="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1682000" > <i>P<sub>kdpF</sub></i></a>, and combined it with a GFP reporter, <a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>, in BioBrick RFC10 standard. The promoter activity was reported by the GFP level under different [K<sup>+</sup>].</p>
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                                        <div class="des">
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<p style="font-size:110%; padding-left:6%;"><strong>Figure 3. K<sup>+</sup> sensing construct with reporter, <a href ="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1682005" >BBa_K1682005</a>.</strong></p></div>
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<p class="subTitle"><i>EcoR</i>I Illegal Site</p>
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<hr class="para">
<p>To make <i>P<sub>kdpF</sub></i> compatible with RFC10 standard and, as so, readily accessible to iGEM community, we designed variants of it with the <i>EcoR</i>I 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 (<a href ="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1682002">BBa_K1682002</a>), C mutant (<a href ="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1682003">BBa_K1682003</a>) and G mutant (<a href ="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1682004">BBa_K1682004</a>) respectively in the following context.
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<p  class="subTitle">Endogenous potassium sensing system in <i>E. coli</i></p>
                                                <div class="project_image" style="padding-top:0">
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<img style="width:80%;" src="https://static.igem.org/mediawiki/2015/8/85/Team_HKUST-Rice_2015_potassium_figure_2.png" alt="image caption">
<img style="width:90%; height:60%;" src="https://static.igem.org/mediawiki/2015/2/2d/HKUST-Rice15_potassium_illegal_site.png" alt="image caption">
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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>
<div class="des">
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<p style="font-size:110%; padding-left:6%;"><strong>Figure 4. Comparison of relative RNAP binding affinity at mutated site of wild type <i>P<sub>kdpF</sub></i> and its variants.</strong></p></div>
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<p  class="subTitle">Interference from other Endogenous Systems</p>
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<p>Other than the inducible KdpFABC system, <i>E. coli</i> has Trk and Kup systems which are constitutively expressed, low-affinity
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potassium transport systems (Epstein & Kim, 1971). Laermann et al. (2013) found that the expression level of <i>KdpFABC</i> is generally
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                                        higher in a strain without the Trk and Kup systems. In other words, the activity of <i>P<sub>kdpF</sub></i> may not be truly reflected due to its
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masking by the Trk and Kup systems. We decided to measure the activity of <i>P<sub>kdpF</sub></i> in <i>E. coli</i> TK2240 (kdp+ Δtrk Δkup) strain, so that we are able to characterize it in a more accurate manner.</p>
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<h1 id="results">Measurement and Characterization</h1>
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<p>To make it more easily used by others, we characterized <i>P<sub>kdpF</sub></i> by relative
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promoter unit (RPU) measurement. Additionally, relative fluorescence measurement has also been done to compare the activities between wild type promoters and 3 variants.</p>
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<p  class="subTitle">Relative promoter unit measurement</p>
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<img id="Kgraph" src="https://static.igem.org/mediawiki/2015/9/90/HKUST-Rice15_%28log_10%29_RPU_of_kdpFp--15%2CT_G-_in_DH10B_-RPU-.png" alt="image caption">
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<p style="font-size:110%; padding-left:6%;"><strong>Figure 5. Relative promoter unit (RPU) of <i>P<sub>kdpF</sub></i>[-15,T>G] at different [K<sup>+</sup>].</strong> 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<sup>+</sup>]. Cells were fixed when OD<sub>600</sub>=0.4. The measurement was carried out using fluorescence-activated cell sorting.  Error bar are presented in SEM.</p></div>
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<p>At the lowest [K<sup>+</sup>], the strength of the G mutant promoter was found to be approximately 0.5 RPU. RPU of the promoter decreases as [K<sup>+</sup>] increases. At 0.025 mM [K<sup>+</sup>], 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<sup>+</sup>]. As expected, <i>P<sub>kdpF</sub></i> is turned off at high [K<sup>+</sup>] due to inhibition of KdpD kinase activity by K<sup>+</sup>.</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  class="subTitle">Relative fluorescence measurement</p>
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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>
<div class="project_image">
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<img id="Kgraph3" src="https://static.igem.org/mediawiki/2015/b/bb/HKUST-Rice_2015_4_promoter_RFU_%2B_GFP_syn_rate.png" alt="image caption">
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<div class="des">
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<p style="font-size:110%; padding-left:6%;"><strong>Figure 6 & 7. Activity of <i>P<sub>kdpF</sub></i> in <i>E. coli</i> DH10B in different [K<sup>+</sup>].</strong>Fluorescence/absorbance versus [K<sup>+</sup>] plot is shown on the left while the GFP synthesis rate versus [K<sup>+</sup>] 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 OD<sub>600</sub>=0.4. 2 other measurements were taken every 15 mins afterwards for GFP synthesis rate. Error bar are presented in SEM.</p></div>
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<p>Both C and G mutants expressed higher fluorescence compared to the wild type and the A mutant. As the [K<sup>+</sup>] went up, the activity of <i>P<sub>kdpF</sub></i> 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<sup>+</sup>], 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<sup>+</sup>], 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 <i>P<sub>kdpF</sub></i> variants (Brewster, 2012). The dynamic range of our promoters is between 0 to 0.1 mM of K<sup>+</sup>.</p>
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<div class="project_image">
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<img id="Kgraph2" src="https://static.igem.org/mediawiki/2015/2/2b/HKUST-Rice15_RFU_of_kdpFp_in_DH10B_and_TK2240.png" alt="image caption">
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<div class="des">
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<p style="font-size:110%; padding-left:6%;"><strong>Figure 8. Comparison between the activities of <i>P<sub>kdpF</sub></i>[-15, T>G] in DH10B and TK2240 strain. </strong>Cells were pre-cultured, washed and sub-cultured as previously described in RPU measurement. Measurement took place when OD<sub>600</sub>=0.4. Error bars are represented as SEM.</p></div>
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<hr class="para">
<p>At [K<sup>+</sup>] lower than 0.0125 mM, the acitivity of G mutant in DH10B was significantly greater than that in TK2240 strain. Activity of <i>P<sub>kdpF</sub></i> in DH10B strain decreased with increasing concentrations, while it remained stable in TK2240. Above 0.05 mM [K<sup>+</sup>], the activity of <i>P<sub>kdpF</sub></i> 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<sup>+</sup> uptake, thereby explaining the higher promoter activity observed beyond 0.05 mM [K<sup>+</sup>].</p>
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<class="subTitle">Design and Testing of potassium sensing Device</p>
<h1 id="results">Future Plan</h1>
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<div class="project_image" style="padding-top:0">
<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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<img style="width:80%" src="https://static.igem.org/mediawiki/2015/d/d8/HKUST_Rice15_potassium_figure_int.png" alt="image caption">
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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>
 
 
<div class="project_row">
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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>
<hr class="para">
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<h2>References</h2>
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  <p style="font-size:125%">Brewster, R. C., Jones, D. L., & Phillips, R. (2012). Tuning promoter strength through RNA polymerase binding site design in <i>Escherichia coli</i>.
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<br><br>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.
+
<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>
+
</div>
+
 
</div>
 
</div>
 +
 +
<div class="project_row">
 +
<p  class="subTitle">Relative Promoter Unit Measurement of <i>P<sub>kdpF</sub></i>[-15,T>G]</p>
 +
<div class="project_image">
 +
<img id="Kgraph" src="https://static.igem.org/mediawiki/2015/9/90/HKUST-Rice15_%28log_10%29_RPU_of_kdpFp--15%2CT_G-_in_DH10B_-RPU-.png" alt="image caption">
 +
<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>
 +
<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>
 +
<hr class="para">
 
</div>
 
</div>
 +
 +
<div class="project_row">
 +
<p  class="subTitle">Interference from other Endogenous Systems</p>
 +
<div class="project_image">
 +
<img style="width:80%" src="https://static.igem.org/mediawiki/2015/4/40/HKUST_Rice15_Comparison_of_kdpFp_in_E_2.png" alt="image caption">
 +
<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>
 +
</div>
 +
<div class="des">
 +
<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>
 +
</div>
 +
</div>
 +
 +
<div class="project_row">
 +
<hr class="para">
 +
<h1>Future Plan</h1>
 +
<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>
 +
</div>
 +
 +
<div class="project_row">
 +
<hr class="para">
 +
<h2>Materials and Methods</h2>
 +
<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>
 +
</div>
 +
 +
<div class="project_row">
 +
<hr class="para">
 +
<h2>References</h2>
 +
<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.
 +
<br><br>International Plant Nutrition Institute. (1998). Functions of Potassium in Plants. <i>Better Crops</i>, 82(3).
 +
<br><br>Keseler et al. (2013), EcoCyc: fusing model organism databases with systems biology, <i>Nucleic Acids Research</i> 41: D605-12.</p>
 +
<h2>References on potassium uptake and regulation systesm in <i>E. coli</i></h2>
 +
<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.
 +
<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.
 +
<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.
 +
<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.
 +
<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.
 +
<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.
 +
<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.
 +
<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.
 +
<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.
 +
<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.
 +
<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>
 +
 +
</div>
 +
</div>
 +
</div>
 
</body>
 
</body>
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</html>
 
</html>
 
{{HKUST-Rice Directory}}
 
{{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.