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

 
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<div id= "page_title"><h1>Potassium Sensor - <i>kdpFp</i></h1></div>
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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/Phosphate_Sensor"><img src="https://static.igem.org/mediawiki/2015/7/7a/HKUST-Rice15_rightarrow.png">
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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">
<p style="color:#5570b0; font-size: 130%"> Phosphate sensor </p></a>
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 +
<p style="color:#5570b0; font-size: 130%"> Potassium sensor - Modeling </p></a>
 
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<div class="project_content">
 
<div class="project_content">
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<div class="project_row">
<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 important roles in plants including osmoregulation,
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<table>
CO<sub>2</sub> regulation, starch synthesis and protein synthesis. Hence, the deficiency of K<sup>+</sup> ion will result
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<tr>
in abnormalities in plant growth and metabolism. It is fundamental to determine its concentration in soil in order to provide
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<td style="width:48.5%">
the proper amount of additional potassium that must be added to the plant by any particular fertilizers.
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<figure>
<p>Our aim is to engineer a Potassium sensor that can detect a range of K<sup>+</sup> concentration in the soil to ensure
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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%;">
the suitable soil condition for the plant fitness. We utilized <i>KdpFp</i>, a promoter located upstream of <i>KdpFABC</i> operon
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</figure>
in <I>Escherichia coli</I> which works under low [K<sup>+</sup>] condition. We put it upstream of a GFP reporter so as to
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</td>
characterize the promoter activity. For simplification, <i>KdpFp</i> will be called potassium promoter in the following context.</p>
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<td style="width:3%">
<p> INSERT BADASS GRAPH HERE</p>
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</td>
 
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<td style="width:48.5%">
<div class="project_image">
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<figure>
<img src="https://static.igem.org/mediawiki/2015/b/be/HKUST-Rice15_Resultsbutton.png" alt="image caption">
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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%">
</div>
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</figure>
                </div>
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</td>
 
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<div class="project_row">
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<hr class="para">
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<td style="width:48.5%">
<h1>Endogenous K sensing system</h1>
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<br>
<p>Badass graph please</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>
<div class="project_image">
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<img src="https://static.igem.org/mediawiki/2015/b/be/HKUST-Rice15_Resultsbutton.png" alt="image caption">
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<p>The potassium ion uptake in <i>Escherichia coli</i> is regulated by several systems under different conditions.  
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<td style="width:48.5%">
The potassium ion transporters, Trk and Kup are constitutively expressed (reference?) while KdpFABC, another
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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>
transporter is activated under limited potassium ion concentration (reference?) and controlled by the KdpDE two-component
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</td>
system (TCS) (Polarek, 1992; Walderhaug, 1992). The TCS consists of KdpD< which is a membrane-bound sensor kinase and KdpE which
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</tr>
is the cytoplasmic response regulator. The autophosphorylation of KdpD transfers a phosporyl group to the KdpE upon low potassium
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</table>
concentration (Voelkner, 1993; Puppe, 1996; Jung ,1997a; Jung, 2000). Phosphorylated KdpE activates <i>kdpFABC</i> operon.</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>
<p>KdpDE TCS is stimulated by both intracellular and extracellular potassium ion  concentration (Jung ,2000; Jung, 2001; Roe, 2000;
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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>
Yan, 2011a; Laermann, 2013). The intracellular and extracellular potassium ion concentration shows an inverse correlation to KdpDE
+
<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>
phosphorylation. Under an increase in potassium ion concentration, KdpD phosphatase activity will be enhanced, causing a decrease in
+
phospho-KdpE and <i>kdpFABC</i> expression. Decreasing potassium ion concentration shows the other way around (Zhang, 2014a; Laermann, 2013).</p>
+
<p>KdpFABC transporter, which depends on ATP, is a high affinity P-Type ATPase potassium ion uptake system (Siebers, 1988; Siebers 1989).  
+
The KdpFABC transporter is specific to potassium ion (Km= 10 μM), magnesium ion (Km= 80 μM), as well as other particular substrates
+
(Siebers, 1988). The KdpFABC complex consists of kdpA, kdpB, kdpC, and kdpF subunits. The kdpA subunit controls the binding as well as
+
transportation of substrate and kdpB subunit pairs the energy. The kdpC subunit keeps kdpA and kdpB together (Buurman95). In addition,
+
the <i>kdpF</i> gene encodes a small polypeptide which stabilize the KdpFABC complex (Altendorf98, Gassel99).
+
</p>
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</div>
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+
<div class="project_row">
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<hr class="para">
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<h1>Design of K+ sensing Device</h1>
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<p>As our potassium-sensing device, we adopt the promoter <i>kdpFp</i> from <i>kdpFABC</i> operon with -28 position of transcription
+
start site relative to start the first gene, <i>kdpF</i>. The -10 and -35 box elements have been mapped which are TACCCT and TTGCGA
+
respectively (Sugiura et al., 1992). The transcription factor, phosphorylated KdpE, binds to the kdpFp at binding site located from
+
-71 to -55 site with reference to the transcription start site (Sugiura et al., 1992; Narayanan et al., 2012). We then combined the
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promoter with a downstream GFP generator, BBa_E0240, using BioBrick RFC 10 standard so that the promoter activity in different potassium
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level can be detected and characterized.</p>
+
 
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<p class="subTitle">Illegal Site</p>
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<p>In order to make our promoters, <i>kdpFp</i>, compatible with Biobrick RFC 10 standard, it should not contain any of the illegal
+
sites (as they belong to the prefix and suffix). However, the promoter contains an EcoRI illegal site from -18 to -12 position.
+
To make the potassium promoter readily accessible to the whole iGEM community, we removed that illegal site by mutating the thymine
+
at -15 position to guanine, cytosine and adenine to give rise to 3 promoter mutants. Also, by removing the illegal site, different
+
mutants of this promoter with different activity levels can be generated. We can choose the one with desirable activity to fit our need.</p>
+
<p>Apart from the wild-type kdpFp (containing illegal site), We designed another 3 mutants which have one base-pair at -15 position with reference to the transcription start site, changed from thymine (T) to either cytosine (C), guanine (G) or adenine (A). For convenience, we denote them as kdpFp(A)/A-mutant, kdpFp(G)/G-mutant and kdpFp(C)/C-mutant respectively. All the mutants, thereby, have their illegal site removed. However, we are expecting different activity from the four promoters due to their difference in binding energy between the promoter and RNA polymerase because of the base-pair changes (Brewster, 2012). Therefore, we characterized all of the promoters later to compare their strength by the mean of fluorescence level and RPU measurements so as to obtain a more comprehensive knowledge in the activity and working range of the 4 kdpFp promoters.</p>
+
 
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<p  class="subTitle">Interference from other Endogenous Systems</p>
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<p>Another concern is the masking of activity of <i>kdpFp</i> by other native constitutive potassium transport systems of <i>E.coli</i>.
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Other than the inducible KdpFABC system(Km=2uM), <i>E. coli</i> has Trk and Kup systems which are constitutively expressed, low-affinity
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potassium transport systems (Km=1.5mM) (reference?). A study has found that the expression level of <i>KdpFABC</i> is generally higher in
+
a strain without Trk and Kup system (Laimins et al., 1981). It is due to the fact that <i>E. coli</i> uses the two saturable transporters,
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Trk and Kup, under normal physiological conditions to uptake extracellular potassium ion. Therefore, only at a very low concentration of
+
intracellular potassium, these two systems can no longer satisfy the need of potassium ion of <i>E. coli</i>.  Hence, the kdpFp will be
+
activated and drive the expression of KdpFABC complex to uptake more K<sup>+</sup>. As the result, the activity of <i>kdpFp</i> may be
+
masked. The activity of this promoter cannot be truly reflected over a range of K+ concentration in the presence of these 2 systems
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(Laermann et al., 2013).</p>
+
<p>We decided to measure the activity of <i>kdpFp</i> in <i>E.coli</i> TK2240 (kdp+ Δtrk Δkup) strain, which is a strain defectve in Trk
+
and Kup system. Such that we are able to characterize <i>kdpFp</i> promoter in a more accurate manner.</p>
+
 
</div>
 
</div>
 
 
 
<div class="project_row">
 
<div class="project_row">
<hr class="para">
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<hr class="para">
<h1>Measurement and Characterization</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>In order to assemble a device that can be widely used by all iGEM community, we characterized <i>kdpFp</i> promoter by using Relative
+
<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>
Promoter Unit (RPU). Additionally, we intended to find the comparison of the activities between different promoters, thus, we also
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measured Relative Fluorescence Unit (RFU) of the 4 potassium promoters.</p>
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<p  class="subTitle">Relative promoter Unit measurement</p>
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<div class="project_image">
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<img src="https://static.igem.org/mediawiki/2015/b/be/HKUST-Rice15_Resultsbutton.png" alt="image caption">
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</div>
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<p>badass rpu graph</p>
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<p class="PICdescription"><b>Figure  . Relative promoter unit (RPU) of <i>kdpFp</i>[-15,T>G] across different concentration of K<sup>+</sup>. </b>The measurement
+
of the activity of G mutant was carried out using fluorescence-activated cell sorting. The strength of the promoter is presented in
+
relative to the strength of standard reference promoter.  Error bar are represented as SEM.</p>
+
<p>From the lowest concentration of potassium ion to 0.025 mM, the strength of the G mutant promoter was found to be about 0.5 RPU and
+
decreasing when concentration increased. After the concentration of potassium ion at 0.025 mM, there was no significant change on the
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RPU values. Strength of G mutant <i>kdpFp</i> at the lowest concentration was about 1.5 times higher than those after 0.025 mM.</p>
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<p>High concentration of potassium ion could repress the expression of <i>kdpFp</i>, due to satisfaction of potassium ion by constitutively
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expressed low-affinity K<sup>+</sup> transporter system, Trk and Kup(Laermann et al., 2013). Therefore the activity of <i>kdpFp</i> promoter,
+
that is represented by the relative fluorescence intensity, at high concentration of potassium ion, should be low and it is shown in our
+
experiments. </p>
+
 
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<p  class="subTitle">Relative fluorescence measurement</p>
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<p>graph graph graph</p>
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<div class="project_image">
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<img src="https://static.igem.org/mediawiki/2015/b/be/HKUST-Rice15_Resultsbutton.png" alt="image caption">
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<img src="https://static.igem.org/mediawiki/2015/b/be/HKUST-Rice15_Resultsbutton.png" alt="image caption">
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</div>
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<p>PAIRED graph to be put here</p>
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</div>
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<div class="project_row">
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<hr class="para">
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<h1>Achievement</h1>
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<p>Our team has finished characterizing all the constructs, including the wild type promoter and 3 mutants controlling the expression of GFP.
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We also contemplated the activity of the promoters over a varying range of K<sup>+</sup> concentration. We had discovered a comparison between
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different promoters and a dynamic relationship between K<sup>+</sup> concentration and the promoters’ activity. Upon different concentration
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of K<sup>+</sup>, Potassium sensor will show different fluorescence level due to distinctive effect of K<sup>+</sup> ion to <i>kdpFp</i>. </p>
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<div class="project_image">
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<img src="https://static.igem.org/mediawiki/2015/b/be/HKUST-Rice15_Resultsbutton.png" alt="image caption">
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</div>
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</div>
+
 
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<div class="project_row">
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<hr class="para">
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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
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sensing part. The Kdp system is composed of two major parts, KdpFABC, a high-affinity potassium transporter as well as two types of regulatory
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proteins, a sensory kinase KdpD and a response regulator KdpE. KdpD and KdpE works together as a two-component system, tracking and responding
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to the intra and extracellular potassium level then interacting with the KdpFABC encoding operon. <i>kdpFABC</i> operon is up-regulated under
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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
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ions favors the phosphorylation of KdpE, which then gives rise to the enhancement of the level of phosphorylated KdpE, and as a result, triggers
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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
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combine the promoter with the downstream GPF generator using biobrick RFC 10 so that the change of the promoter activity in different potassium
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level can be detected and characterized.
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</p>
+
<div class="project_image">
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<img src="https://static.igem.org/mediawiki/2015/b/be/HKUST-Rice15_Resultsbutton.png" alt="image caption">
+
</div>
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</div>
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+
<div class="project_row">
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<hr class="para">
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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
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promoter <i>kdpFp</i> contains the EcoRI illegal site. While the second concern is about the background noise contributed by other native
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constitutive potassium transport systems of <i>E. coli</i>, including trk and Kup systems, which are potassium ions influx systems and are
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expected to lower the activity of our promoter <i>kdpFp</i>. </p>
+
<div class="project_image">
+
<img src="https://static.igem.org/mediawiki/2015/b/be/HKUST-Rice15_Resultsbutton.png" alt="image caption">
+
</div>
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</div>
+
 
+
 
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<div class="project_row">
+
<hr class="para">
+
<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.
+
</p>
+
<div class="project_image">
+
<img src="https://static.igem.org/mediawiki/2015/b/be/HKUST-Rice15_Resultsbutton.png" alt="image caption">
+
</div>
+
</div>
+
 
+
 
+
<div class="project_row">
+
<hr class="para">
+
<h1>Result obtained</h1>
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<p class="subTitle">FACS</p>
+
<div class="project_image">
+
<img src="https://static.igem.org/mediawiki/2015/b/be/HKUST-Rice15_Resultsbutton.png" alt="image caption">
+
</div>
+
<p class="PICdescription"><b>Figure  . Relative promoter unit (RPU) of <i>kdpFp</i>[-15,T>G] across different concentration of K+. </b>The measurement of the activity of
+
G mutant was carried out using fluorescence-activated cell sorting. The strength of the promoter is presented in relative to the strength of
+
standard reference promoter.  Error bar are represented as SEM.
+
</p>
+
<p>From the lowest concentration of potassium ion to 0.025 mM, the strength of the G mutant promoter was found to be about 0.5 RPU and decreasing
+
when concentration increased. After the concentration of potassium ion at 0.025 mM, there was no significant change on the RPU values. Strength of
+
G mutant <i>kdpFp</i> at the lowest concentration was about 1.5 times higher than those after 0.025 mM.
+
</p>
+
<p>High concentration of potassium ion could repress the expression of <i>kdpFp</i>, due to satisfaction of potassium ion by constitutively expressed
+
low-affinity K<sup>+</sup> transporter system, Trk and Kup(Laermann et al., 2013). Therefore the activity of <i>kdpFp</i> promoter, that is represented
+
by the relative fluorescence intensity, at high concentration of potassium ion, should be low and it is shown in our experiments.
+
</p>
+
 
 
<p class="subTitle">GFP synthesis rate and RFU</p>
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<hr class="para">
<div class="project_image">
+
<p class="subTitle">Endogenous potassium sensing system in <i>E. coli</i></p>
<img src="https://static.igem.org/mediawiki/2015/b/be/HKUST-Rice15_Resultsbutton.png" alt="image caption">
+
<img style="width:80%;" src="https://static.igem.org/mediawiki/2015/8/85/Team_HKUST-Rice_2015_potassium_figure_2.png" alt="image caption">
</div>
+
<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 class="PICdescription"><b>Figure  . Activity of <i>kdpFp</i> in <i>E. coli</i> DH10B in different K<sup>+</sup> concentrations.</b>A, T C and G represent A mutant, wild
+
type promoter, C mutant and G mutant respectively. Error bar are represented as SEM.
+
</p>
+
<p>Both C and G mutants similarly expressed higher fluorescence intensity compared to the wild type and the A mutant. A higher level of potassium ion
+
inside K minimal medium repressed the expression level of <i>kdpFp</i>, which was shown by lower fluorescence intensity of all promoters at higher
+
concentration of potassium ion. The expression level of both C and G mutant promoters were significantly higher than the wild type one, while A mutant
+
promoter was always the lowest.
+
</p>
+
<p>The fluorescence intensity decreases over the concentration of potassium ion in K minimal medium. After several trials, we found the dynamic range
+
of our promoters. It is between 0 to 0.1 mM of potassium ion in K minimal medium. Thus, we characterized our promoters at those range of concentrations
+
(0 to 0.2 mM).
+
</p>
+
<p>We observed that the fluorescence intensity expressed by C and G mutants are always at around similar values since the binding affinity might be
+
the same with reference to the energy matrix (reference?). Moreover, both of them are always higher than the one expressed by the wild type promoter.
+
Meanwhile, the fluorescence intensity expressed by A mutant is lower than the the one expressed by the wild-type promoter. Also, the relationship of
+
relative fluorescence intensity and K<sup>+</sup> concentration of all the promoters are  coherent with the previous RPU measurement of G mutant.
+
</p>
+
 
+
<div class="project_image">
+
<img src="https://static.igem.org/mediawiki/2015/b/be/HKUST-Rice15_Resultsbutton.png" alt="image caption">
+
</div>
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<p class="PICdescription"><b>Figure . The activities of kdpFp in the medium containing no potassium ion.</b>A fluorescence/absorbance plot was obtained from measuring the relative fluorescence level exhibited by the promoters, <i>kdpFp</i> and its 3
+
mutants (A, C and G), in DH10B cells in 0 mM K minimal medium. The expression level of both C and G mutants are significantly higher than the expression
+
level of wild-type promoter and A mutant promoter. There was no significant difference between the activity of G mutant and C mutant promoters,
+
as well as between wild type promoter and A mutant. Error bars are represented as SEM.
+
</p>
+
<p>We also would like to make a clear comparison between the expression level of the different mutant promoters. We tried to make a comparison
+
of the expression of all the promoters at the lowest level of potassium ion, that is no potassium ion in K minimal medium. From the graph that we
+
obtained, we observed that the expression level of both C and G mutants are about 2 times higher than the wild type promoter and A mutant. These
+
results demonstrate our former experiments that the relative fluorescence intensity level of both C and G mutants are always higher than both
+
wild type promoter and A mutant.
+
</p>
+
 
 
<div class="project_image">
+
<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.
<img src="https://static.igem.org/mediawiki/2015/b/be/HKUST-Rice15_Resultsbutton.png" alt="image caption">
+
</div>
+
<p class="PICdescription"><b>Figure  . Comparison between the activities of kdpFp[-15, T>G] in DH10B and TK2240 strain. </b>A fluorescence/absorbance plot was obtained from measuring the relative fluorescence level exhibited by the G mutant pormoter in DH10B and  
+
TK2240 cells. At the concentration of potassium ion lower than 0.0125 mM, the fluorescence intensity expressed by G mutant in DH10B was significantly
+
greater than the one in TK2240 strain. The fluorescence intensity expressed by G mutant in DH10B strain decreased over the concentration, while the
+
one in TK2240 remained the same. Starting from the concentration of potassium ion at 0.0125 mM onwards, the difference of GFP expressed by both strains
+
was not significant. After passing the concentration of potassium ion at 0.05 mM, the expression level of kdpFp in TK2240 strain exceeded the one
+
in the DH10B strain. Error bars are represented as SEM.
+
</p>
+
<p>We did another relative fluorescnece measurement using TK2240 to observe a more accurate  activity of kdpFp. The TK2240 strain is defective
+
in trk and kup gene. The only gene which could respond to potassium ion in TK2240 strain is only kdp gene. Therefore the expression of kdp gene
+
would not be masked by the saturable low-affinity K<sup>+</sup> transporter system. We were expecting that the activity of <i>kdpFp</i> in TK2240 are
+
higher than that in DH10B. The fact mentioned earlier corresponds to the graph at higher concentration of potassium ion, although the expression
+
level in DH10B is higher than in TK2240 at low concentration of potassium ion.
+
</p>
+
<p>We could observe the difference of the fluorescence intensity expressed by both strains at the concentration of potassium ion lower than 0.0125 mM.
+
The fluorescence intensity expressed by G mutant in DH10B strain decreases over the concentration, while the one in TK2240 remains the same. Since
+
TK2240 has a greater need of kdpFABC complex to help scavenge the external K+, so what might have caused this would be TK2240 spend too much energy
+
at that stage to make kdpFABC complex and thus spare less energy on producing GFP.
+
</p>
+
<p>At higher concentration of potassium ion, starting from 0.0125 mM onwards, the difference of fluorescence intensity expressed by both strains
+
is not significant anymore. Both strains show decrease in fluorescence intensity over the increasing concentration of potassium ion in K minimal medium.
+
</p>
+
<p>After passing the concentration of potassium ion at 0.05 mM, the fluorescence intensity expressed by TK2240 strain exceeds the one in the DH10B
+
strain due to the constant need of potassium ion of the cells and in TK2240, there is only KdpFABC transporter system that can satisfy this need of <i>E. coli</i>.
+
</p>
+
 
+
<div class="project_image">
+
<img src="https://static.igem.org/mediawiki/2015/b/be/HKUST-Rice15_Resultsbutton.png" alt="image caption">
+
</div>
+
<p class="PICdescription"><b>Figure . The GFP synthesis rate of kdpFp mutants in DH10B. </b>A GFP synthesis rate plot was obtained from measuring the relative fluorescence level exhibited by the <i>kdpFp</i> and its mutants in DH10B cells.
+
From the lowest concentration of potassium ion to 0.025 mM, there was no significant change of GFP synthesis rate. The GFP rate for C and G mutant
+
promoters were always higher than the wild type and A mutant promoters. Starting from the concentration of potassium ion at 0.025 mM, the values
+
of GFP synthesis rate dropped to nearly 0. After the concentration of potassium ion reached 0.1 mM, the GFP synthesis rate became so low and there
+
was no significant differences for all mutants.
+
</p>
+
<p>The GFP synthesis rate was calculated by measuring the rate of fluorescence intensity level over time. As it can be seen from the GFP synthesis
+
rate graph, there was no significant difference of the GFP synthesis rate at low concentration of potassium ion until 0.025 mM. From the results
+
that we always obtained, the rate for C and G mutant promoters were always greater than the wild-type and A mutant promoters. This explained
+
why the expression level of C and G mutants were higher than the wild type and A mutant.
+
</p>
+
<p>When the concentration of potassium ion was higher than 0.025 mM of potassium ion in K minimal medium, the values of GFP synthesis rate
+
dropped to nearly 0. This conveyed that the fluorescence intensity started to decrease significantly at 0.025 mM potassium ion concentration
+
onwards.
+
</p>
+
<p>
+
Finally, at 0.1 mM, the GFP synthesis rate values were so low, the relative fluorescence intensity, therefore, would also be so low.
+
</p>
+
+
<p class="subTitle">Considerations for replicating the experiments</p>
+
<p>a. OD referring to mid log phase of e coli</p>
+
<p>b. dilution at the beginning</p>
+
<p style="padding-left:2em">i. We do measurement when the cells are in the mid log phase. As stated from the paper, <i>E. coli</i> enters mid
+
log phase when the value of OD600 is between 0.3 to 0.5. Therefore to have all cultures entering the log phase together by the time we are
+
doing the measurement, we always do dilution to make the OD values around the same before we incubate the cultures inside the 96 deep well
+
plates. We believe that the growth of the cells will be similar if the starting conditions are the same, which we think are the OD values.
+
 
</p>
 
</p>
 +
<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>
 
 
 +
<hr class="para">
 
</div>
 
</div>
 
 
 +
<div class="project_row">
 +
<p  class="subTitle">Design and Testing of potassium sensing Device</p>
 +
<div class="project_image" style="padding-top:0">
 +
<img style="width:80%" src="https://static.igem.org/mediawiki/2015/d/d8/HKUST_Rice15_potassium_figure_int.png" alt="image caption">
 +
<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>
 +
<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>
 +
 +
<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">
 
</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 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>
 
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+
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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.