Difference between revisions of "Team:Bielefeld-CeBiTec/Accumulation"

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Accumulation

Short Summary

Increasing the accumulation of copper ions in living cells requires enhanced copper uptake as well as inhibition of copper export systems. We knocked out cusCFBA, the dominant copper export system from Escherichia coli which facilitates Cu(I) export from the cytoplasm out of the cell. In addition, we supplemented the native copper accumulation system of E. coli by expression of the outer membrane importers copC and oprC as well as the inner membrane transporters copD and hmtA.

Introduction

One important objective of nanoFactory is to construct an efficient accumulation system for heavy metal ions in order to subsequently form nanoparticles in E. coli. As a proof of principle we concentrated on the accumulation of Cu(II) ions. Besides the knockout of copper exporters, the main component for such a system is a highly efficient uptake system.

Since copper is toxic to all types of cells (Ladomersky & Petris, 2015), the existence of transporters for the active and selective uptake of copper ions may initially come as a surprise, but copper also carries out important functions in many cells. As a cofactor for many different enzymes such as superoxide dismutase it is primarily involved in electron transfer, dioxygen transport and activation (Solomon et al., 2014). Accordingly, there must be natural absorption mechanisms for the trace element both into the periplasm and the cytoplasm. Nevertheless, as non-bonded, dissolved copper in particular is very toxic, there are efficient export systems in E. coli to keep a tightly controlled copper homeostasis and remove excess copper from the cell (Rensing & Grass, 2003). In order to create an efficient accumulation system for copper ions, highly selective uptake systems must be expressed on the one hand and existing export systems must be suppressed on the other.

**Figure 1:** Overview on the different copper accumulation components we worked with. The active copper export system cusCBA is knocked out and prevents Cu(I) loss. Uptake is promoted from the outer membrane importer OprC supported by the inner membrane importers CopD and HmtA. CopC is a metallochaperone delivering Cu(II) to CopD. Toxic Cu(I) ions in the periplasm are oxidated to Cu(II) in the periplasm by the native copper oxidase Cue

Preventing Copper Export

In order to prevent the loss of the effortfully imported Cu(II) ions by transport or diffusion, the cus operon of E. coli was knocked out using CRISPR/Cas9. It consists of cusCFBA which belongs to the resistance-nodulation-cell division superfamily (Tseng et al., 1999) and the two-component regulatory system cusRS. The response regulator cusR and its associated membrane-bound kinase cusS regulate the expression of the opposed directed cusCFBA genes directly upstream of cusRS (Munson et al., 2000, Xiao et al., 2017). The export of Cu(I) and also Ag(I) is carried out by the tripartite protein complex CusCBA (Gudipaty et al., 2012) which is spanning through both the inner and outer membrane of the cell (Delmar, Su & Yu, 2013). CusA is a pump for Cu(I)//Ag(I) ions located in the inner cell membrane. It pumps Cu(I)/Ag(I) ions, driven by proton-motive force, through the adapter-like CusB and CusC which span the outer membrane (Franke et al., 2003, Delmar, Su & Yu, 2013). Cu(I)/Ag(I) ions from the periplasmic space are delivered to CusB by CusF metallochaperones (Bagai et al., 2008).

We constructed a knock out mutant of E. coli DH5α using CRISPR/Cas9 to remove the whole cus operon ultimately prohibiting Cu(I) export. Consequently Cu(I) ions can not leave the periplasmic space. Subsequently the oxidization of Cu(I) to Cu(II) occurs spontaneously in the oxidative environment of aerobically grown cultures and is catalyzed by multicopper oxidase CueO which is native to E. coli (Outten et al., 2001).

**Figure 2:** Structure of the *E. coli cus* operon. The two-component regulatory system *cusRS* is located directly downstream of the *cusCFBA* coding sequences.

Copper Import

The absorption of copper takes place in two steps: In the gram-negative chassis organism E. coli, Cu(II) ions are first transported across the outer cell membrane into the periplasm and subsequently into the cytoplasm over the inner cell membrane if necessary. Due to its toxicity an accumulation of copper ions in the periplasm has many advantages over accumulating it in the cytoplasm, where damage to the DNA and several proteins might occur (Ladomersky & Petris, 2015).

As comparable uptake system are not known in E. coli (Rensing & Grass, 2003) we identified the outer membrane porin OprC from Pseudomonas aeruginosa as a good candidate for this purpose (Yoneyama & Nakae, 2001). OprC belongs to the superfamily of TonB-dependent receptors to which TonB binds as a signal protein (Postle & Good, 1983). It was shown that OprC allows the selective uptake of Cu(I/II) into the periplasmic space whilst other toxic ions like Ag(I) and Hg(II) are not imported (Yoneyama & Nakae, 1996, Quintana, Novoa-Aponte & Argüello, 2017). For our project we used a OprC homolog of P. brassicacearum 3Re2-7 (strain not pulished yet, for more information please contact Prof. Dr. Alfred Pühler) with 71% protein identity to avoid working with an organism from risk group two.

In order to further increase the intracellular concentration of copper ions, the P-type ATPase HmtA from P. aeruginosa can be used. It actively transports copper ions across the cytoplasmic membrane into the cytoplasm (Lewinsohn, Lee & Rees, 2009). As with oprC the homolog from the non-pathogenic P. brassicacearum 3Re2-7 with a protein identity of 80% was used. HmtA expression was found to lead to acute hypersensitivity for Cu(II) and Zn(II), which is a result of Cu(II)/Zn(II) uptake consequently leading to increased intracellular concentrations of these metal ions (Lewinsohn, Lee & Rees, 2009). Uptake of other cations into the cytoplasm like toxic Ag(I) and Cd(II) ions is not facilitated by hmtA expression (Lewinson, Lee & Rees, 2009). As hmtA expression depends on the extracellular Zn(II) concentration and Zn(II) is transported as well as Cu(II) by HmtA (Pederick et al., 2015), it can be assumed that HmtA is a zinc importer which also transports copper ions due to the similarity in size and charge of Zn(II) and Cu(II) ions.

Another transportation system is copCD from the copABCD operon for copper homeostasis in Pseudomonas syringae pv. tomato. This operon encodes for a precise regulation system of intracellular Cu(II) concentration (Lawton et al., 2016). The two genes copC and copD exist in various forms like in a copABCD operon, standing alone as a copCD operon or even build up CopCD fusion proteins (Arnesano et al., 2002, Lawton et al., 2016) and their function is not fully understood yet. One proposed function is the uptake of Cu(II) ions into the cytoplasm (Cha & Cooksey, 1993). CopC is a putative metallochaperone and exists in different forms with one or two copper binding sites (Lawton et al., 2016) and CopD is a inner membrane protein that is presumably able to pump Cu(II) into the cell. We identified both genes in the genome of P. brassicacearum 3Re2-7 and transferred them into E. coli to increase the uptake of Cu(II) and accumulate intracellular copper.

Conclusion

Our copper accumulation system works in two ways. We prevent the export of Cu(I) by knocking out the native E. coli copper export cus operon. In addition we identified genes involved in copper uptake, hmtA, oprC, copC and copD, and cloned them into E. coli. This combined system provides and represents an highly efficient and specific accumulation system for copper ions.

The results of the accumulation experiments can be found here.

Arnesano, F., Banci, L., Bertini, I., & Thompsett, A. R. (2002). Solution structure of CopC: a cupredoxin-like protein involved in copper homeostasis. Structure, 10(10), 1337-1347.
Bagai, I., Rensing, C., Blackburn, N. J., & McEvoy, M. M. (2008). Direct metal transfer between periplasmic proteins identifies a bacterial copper chaperone. Biochemistry, 47(44), 11408-11414.
Cha, J. S., & Cooksey, D. A. (1993). Copper hypersensitivity and uptake in Pseudomonas syringae containing cloned components of the copper resistance operon. Applied and environmental microbiology, 59(5), 1671-1674.
Delmar, J. A., Su, C. C., & Edward, W. Y. (2013). Structural mechanisms of heavy-metal extrusion by the Cus efflux system. Biometals, 26(4), 593-607.
Franke, S., Grass, G., Rensing, C., & Nies, D. H. (2003). Molecular analysis of the copper-transporting efflux system CusCFBA of Escherichia coli. Journal of bacteriology, 185(13), 3804-3812.
Gudipaty, S. A., Larsen, A. S., Rensing, C., & McEvoy, M. M. (2012). Regulation of Cu (I)/Ag (I) efflux genes in Escherichia coli by the sensor kinase CusS. FEMS microbiology letters, 330(1), 30-37.
Ladomersky, E., & Petris, M. J. (2015). Copper tolerance and virulence in bacteria. Metallomics, 7(6), 957-964.
Lawton, T. J., Kenney, G. E., Hurley, J. D., & Rosenzweig, A. C. (2016). The CopC family: structural and bioinformatic insights into a diverse group of periplasmic copper binding proteins. Biochemistry, 55(15), 2278-2290.
Lewinson, O., Lee, A. T., & Rees, D. C. (2009). A P-type ATPase importer that discriminates between essential and toxic transition metals. Proceedings of the National Academy of Sciences, 106(12), 4677-4682.
McDevitt, C. A. (2015). ZnuA and zinc homeostasis in Pseudomonas aeruginosa. Scientific reports, 5, 13139.
Munson, G. P., Lam, D. L., Outten, F. W., & O'Halloran, T. V. (2000). Identification of a copper-responsive two-component system on the chromosome of Escherichia coli K-12. Journal of Bacteriology, 182(20), 5864-5871.
Outten, F. W., Huffman, D. L., Hale, J. A., & O'Halloran, T. V. (2001). The independent cue and cusSystems confer copper tolerance during aerobic and anaerobic growth inEscherichia coli. Journal of Biological Chemistry, 276(33), 30670-30677.
Pederick, V. G., Eijkelkamp, B. A., Begg, S. L., Ween, M. P., McAllister, L. J., Paton, J. C., & McDevitt, C. A. (2015). ZnuA and zinc homeostasis in Pseudomonas aeruginosa. Scientific reports, 5, 13139.
Postle, K., & Good, R. F. (1983). DNA sequence of the Escherichia coli tonB gene. Proceedings of the National Academy of Sciences, 80(17), 5235-5239.
Quintana, J., Novoa-Aponte, L., & Argüello, J. M. (2017). Copper homeostasis networks in the bacterium Pseudomonas aeruginosa. Journal of Biological Chemistry, jbc-M117.
Rensing, C., & Grass, G. (2003). Escherichia coli mechanisms of copper homeostasis in a changing environment. FEMS microbiology reviews, 27(2-3), 197-213.
Solomon, E. I., Heppner, D. E., Johnston, E. M., Ginsbach, J. W., Cirera, J., Qayyum, M., ... & Tian, L. (2014). Copper active sites in biology. Chemical reviews, 114(7), 3659-3853.
Tseng, T. T., Gratwick, K. S., Kollman, J., Park, D., Nies, D. H., Goffeau, A., & Saier Jr, M. H. (1999). The RND permease superfamily: an ancient, ubiquitous and diverse family that includes human disease and development proteins. Journal of molecular microbiology and biotechnology, 1(1), 107-125.
Xiao, M., Zhu, X., Fan, F., Xu, H., Tang, J., Qin, Y., ... & Zhang, X. (2017). Osmotolerance in Escherichia coli is improved by activation of copper efflux genes or supplementation with sulfur containing amino acids. Applied and environmental microbiology, AEM-03050.
Yoneyama, H., & Nakae, T. (1996). Protein C (OprC) of the outer membrane of Pseudomonas aeruginosa is a copper-regulated channel protein. Microbiology, 142(8), 2137-2144.

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@@ Line 36: / Line 56: @@
 <body style="height:100%;">
+		<div class="title_picture">
+			<img src="https://static.igem.org/mediawiki/2018/e/e7/T--Bielefeld-CeBiTec--kolben_vk.png" style="width:100%">
+		</div>
+ <div class="sidenavi" id="side_bar">
+                        <li class="side_list">
+			<a href="#intro">Introduction</a>
+			</li>
+			<li class="side_list">
+			<a href="#prev">Preventing Copper Export</a>
+			</li>
+			<li class="side_list">
+			<a href="#copim">Copper Import</a>
+			</li>
+			<li class="side_list">
+			<a href="#con">Conclusion</a>
+			</li>
+		</div>
        <div class="container">
             <div class="main_content">
+                   <div class="title">Accumulation</div>
-                 <div class="title">Copper Accumulation System</div>
+<h2><span id="exp"></span>Short Summary</h2>
-		  <article>
+<article>Increasing the accumulation of copper ions in living cells requires enhanced copper uptake as well as inhibition of copper export systems. We knocked out <i>cusCFBA</i>, the dominant copper export system from <i>Escherichia coli</i> which facilitates Cu(I) export from the cytoplasm out of the cell. In addition, we supplemented the native copper accumulation system of <i>E. coli</i> by expression of the outer membrane importers <i>copC</i> and <i>oprC</i> as well as the inner membrane transporters <i>copD</i> and <i>hmtA</i>.
-                  </article>
+</article>
-                   <h2>Overview</h2>
+<a name="intro" id="intr" class="shifted-anchor"></a>
-                   <br><br>
+<h2>Introduction</h2>
-                   <article>
-One important goal of nanoFactory is to create an efficient accumulation system for heavy metal                         ions, in this case of copper, in order to subsequently form nanoparticles in <i>Escherichia coli</i>. The main component for such a system is a highly efficient uptake system.
+<article>
+One important objective of nanoFactory is to construct an efficient accumulation system for heavy metal ions in order to subsequently form nanoparticles in <i>E. coli</i>. As a proof of principle we concentrated on the accumulation of Cu(II) ions. Besides the knockout of copper exporters, the main component for such a system is a highly efficient uptake system.
 <br><br>
-Since copper is toxic to all types of cells (Ladomersky & Petris, 2015), the presence of transporters for the active and selective uptake of copper ions may initially come as a surprise. But copper also carries out important functions in many cells. As a cofactor for many different enzymes such as superoxide dismutase, it is primarily involved in electron transfer and dioxygen transport and activation (Solomon <i>et al.</i>, 2014). Accordingly, there must be natural absorption mechanisms for this trace element into both the periplasm and the cytoplasm. Nevertheless non-bonded, dissolved copper in particular is very toxic, so there are also efficient export systems in <i>E. coli</i> to get rid of excess copper (Rensing & Grass, 2003). In order to create an efficient accumulation system for copper ions, efficient and selective uptake systems must be expressed on the one hand and existing export systems must be suppressed on the other.
+Since copper is toxic to all types of cells (Ladomersky & Petris, 2015), the existence of transporters for the active and selective uptake of copper ions may initially come as a surprise, but copper also carries out important functions in many cells. As a cofactor for many different enzymes such as superoxide dismutase it is primarily involved in electron transfer, dioxygen transport and activation (Solomon <i>et al.</i>, 2014). Accordingly, there must be natural absorption mechanisms for the trace element both into the periplasm and the cytoplasm. Nevertheless, as non-bonded, dissolved copper in particular is very toxic, there are efficient export systems in <i>E. coli</i> to keep a tightly controlled copper homeostasis and remove excess copper from the cell (Rensing & Grass, 2003). In order to create an efficient accumulation system for copper ions, highly selective uptake systems must be expressed on the one hand and existing export systems must be suppressed on the other.</article>
-                    </article>
+<br><br>
+<figure role="group">
+                      <img class="figure eighty" src="https://static.igem.org/mediawiki/2018/8/8a/T--Bielefeld-CeBiTec--ES--Accumulation-4-Importer.png">
+                      <figcaption>
+                           <b><br>Figure 1:</b> Overview on the different copper accumulation components we worked with. The active copper export system cusCBA is knocked out and prevents Cu(I) loss. Uptake is promoted from the outer membrane importer OprC supported by the inner membrane importers CopD and HmtA. CopC is a metallochaperone delivering Cu(II) to CopD. Toxic Cu(I) ions in the periplasm are oxidated to Cu(II) in the periplasm by the native copper oxidase Cue
+                      </figcaption>
+                    </figure>
-                   <h2>Import</h2>
-                   <article>
+<a name="prev" id="prev" class="shifted-anchor"></a>
-                   The absorption of copper takes place in two steps: In the gram-negative chassis organism <i>E. coli</i>, copper is first transported across the outer cell membrane into the periplasm and transported to the cytoplasm over the inner cell membrane if further needed. Due to its toxicity an accumulation of copper in the periplasm has many advantages over accumulating it in the cytoplasm, where DNA damage might be a problem (Ladomersky & Petris, 2015). For this purpose, the transport of copper into the periplasm, the porin OprC from <i>Pseudomonas brassicacearum</i> is used, as comparable uptake systems in <i>E. coli</i> are not known (Rensing & Grass, 2003). In the closely related pathogenic organism <i>Pseudomonas aeruginosa</i> (71% protein identity) it could be shown that OprC allows for a selective uptake of Cu(II) ions along a concentration gradient, while toxic ions such as Ag(I), Cu(I) or Hg(II) are held back (Yoneyama & Nakae, 1996). OprC belongs to the superfamily of TonB-dependent receptors to which TonB binds as a signal protein (Postle & Good, 1983).
+<h2>Preventing Copper Export</h2>
-                   <br><br>
+<article>In order to prevent the loss of the effortfully imported Cu(II) ions by transport or diffusion, the <i>cus</i> operon of <i>E. coli</i> was knocked out using CRISPR/Cas9. It  consists of <i>cusCFBA</i> which belongs to the resistance-nodulation-cell division superfamily (Tseng <i>et al.</i>, 1999) and the two-component regulatory system <i>cusRS</i>. The response regulator <i>cusR</i> and its associated membrane-bound kinase <i>cusS</i> regulate the expression of the opposed directed <i>cusCFBA</i> genes directly upstream of <i>cusRS</i> (Munson <i>et al.</i>, 2000, Xiao <i>et al.</i>, 2017). The export of Cu(I) and also Ag(I) is carried out by the tripartite protein complex CusCBA (Gudipaty <i>et al.</i>, 2012) which is spanning through both the inner and outer membrane of the cell (Delmar, Su & Yu, 2013). CusA is a pump for Cu(I)//Ag(I) ions located in the inner cell membrane. It pumps Cu(I)/Ag(I) ions, driven by proton-motive force, through the adapter-like CusB and CusC which span the outer membrane (Franke <i>et al.</i>, 2003, Delmar, Su & Yu, 2013). Cu(I)/Ag(I) ions from the periplasmic space are delivered to CusB by CusF metallochaperones (Bagai <i>et al.</i>, 2008).
-In order to increase the concentration of copper ions further, the P-type ATPase HmtA of <i>P. aeruginosa</i> can be used. It actively transports copper ions across the cytoplasmic membrane into the cytoplasm (Lewinsohn, Lee & Rees, 2009). As with the <i>OprC</i>-gene the homologue from the non-pathogenic <i>P. brassicacearum</i> with a protein identity of 80% is used. <i>HmtA</i> expression was found to result in acute hypersensitivity for Cu(II) and Zn(II), which is a result of Cu(II)/Zn(II) uptake, as the expression resulted in increased intracellular concentration of these metal ions (Lewinsohn, Lee & Rees, 2009). Uptake of other cations into the cytoplasm like toxic Ag(I) and Cd(II) ions did not occur (Lewinsohn, Lee & Rees, 2009). As <i>HmtA</i> expression depends on the extracellular Zn(II) concentration and Zn(II) is transported as well as Cu(II) by the HmtA-transporter (Pederick <i>et al.</i>, 2015), it can be assumed that HmtA is a zinc importer, which also transports copper ions due to the similarity of size and charge of Zn(II) and Cu(II) ions.
+<br><br>
-                   </article>
+We constructed a knock out mutant of <i>E. coli</i> DH5α using CRISPR/Cas9 to remove the whole <i>cus</i> operon ultimately prohibiting Cu(I) export. Consequently Cu(I) ions can not leave the periplasmic space. Subsequently the oxidization of Cu(I) to Cu(II) occurs spontaneously in the oxidative environment of aerobically grown cultures and is catalyzed by multicopper oxidase CueO which is native to <i>E. coli</i> (Outten <i>et al.</i>, 2001).</article>
-                   <h2>Export</h2>
-                   <article>
-                   In order to prevent the loss of imported Cu(II) ions by active or passive export, the genes <i>CusABCFRS</i> from copper homeostasis were knocked out with CRISPR/Cas9. CusR and CusS together build up a two-component system, which regulates the expression of the <i>cusCFBA</i> operon. (Xiao <i>et al.</i>, 2017). <i>CusCFBA</i> genes encode for Ag(I)- and Cu(I)-exporting efflux proteins.
-                   </article>
-<!--
+<figure role="group">
-                    <figure role="group">
+                       <img class="figure eighty" src="https://static.igem.org/mediawiki/2018/5/53/T--Bielefeld-CeBiTec--ES--cus-Operon.png">
-                       <img class="figure eighty" src="https://static.igem.org/mediawiki/2018/f/f3/T--Bielefeld-CeBiTec--800pxMOsafety2.jpg">
                        <figcaption>
-                            <b>Figure X:</b>
+                            <b><br>Figure 2:</b> Structure of the <i>E. coli cus</i> operon. The two-component regulatory system <i>cusRS</i> is located directly downstream of the <i>cusCFBA</i> coding sequences.
                        </figcaption>
                     </figure>
-		<table id="t01" class="centern">
+<a name="copim" id="copim" class="shifted-anchor"></a>
-		  <tr>
+<h2>Copper Import</h2>
-			<th>Firstname</th>
+<article>The absorption of copper takes place in two steps: In the gram-negative chassis organism <i>E. coli</i>, Cu(II) ions are first transported across the outer cell membrane into the periplasm and subsequently into the cytoplasm over the inner cell membrane if necessary. Due to its toxicity an accumulation of copper ions in the periplasm has many advantages over accumulating it in the cytoplasm, where damage to the DNA and several proteins might occur (Ladomersky & Petris, 2015).
-			<th>Lastname</th>
+<br><br>
-			<th>Age</th>
+As comparable uptake system are not known in <i>E. coli</i> (Rensing & Grass, 2003) we identified the outer membrane porin OprC from <i>Pseudomonas aeruginosa</i> as a good candidate for this purpose (Yoneyama & Nakae, 2001). OprC belongs to the superfamily of TonB-dependent receptors to which TonB binds as a signal protein (Postle & Good, 1983). It was shown that OprC allows the selective uptake of Cu(I/II) into the periplasmic space whilst other toxic ions like Ag(I) and Hg(II) are not imported (Yoneyama & Nakae, 1996, Quintana, Novoa-Aponte & Argüello, 2017). For our project we used a OprC homolog of <i>P. brassicacearum</i> 3Re2-7 (strain not pulished yet, for more information please contact Prof. Dr. Alfred Pühler) with 71% protein identity to avoid working with an organism from risk group two.
-		  </tr>
+<br><br>
-		  <tr>
+In order to further increase the intracellular concentration of copper ions, the P-type ATPase HmtA from <i>P. aeruginosa</i> can be used. It actively transports copper ions across the cytoplasmic membrane into the cytoplasm (Lewinsohn, Lee & Rees, 2009). As with <i>oprC</i> the homolog from the non-pathogenic <i>P. brassicacearum</i> 3Re2-7 with a protein identity of 80% was used. <i>HmtA</i> expression was found to lead to acute hypersensitivity for Cu(II) and Zn(II), which is a result of Cu(II)/Zn(II) uptake consequently leading to increased intracellular concentrations of these metal ions (Lewinsohn, Lee & Rees, 2009). Uptake of other cations into the cytoplasm like toxic Ag(I) and Cd(II) ions is not facilitated by <i>hmtA</i> expression (Lewinson, Lee & Rees, 2009). As <i>hmtA</i> expression depends on the extracellular Zn(II) concentration and Zn(II) is transported as well as Cu(II) by HmtA (Pederick <i>et al.</i>, 2015), it can be assumed that HmtA is a zinc importer which also transports copper ions due to the similarity in size and charge of Zn(II) and Cu(II) ions.
-			<td>Jill</td>
+<br><br>
-			<td>Smith</td>
+Another transportation system is <i>copCD</i> from the <i>copABCD</i> operon for copper homeostasis in <i>Pseudomonas syringae</i> pv. <i>tomato</i>. This operon encodes for a precise regulation system of intracellular Cu(II) concentration (Lawton <i>et al.</i>, 2016). The two genes <i>copC</i> and <i>copD</i> exist in various forms like in a <i>copABCD</i> operon, standing alone as a <i>copCD</i> operon or even build up CopCD fusion proteins (Arnesano <i>et al.</i>, 2002, Lawton <i> et al.</i>, 2016) and their function is not fully understood yet. One proposed function is the uptake of Cu(II) ions into the cytoplasm (Cha & Cooksey, 1993). CopC is a putative metallochaperone and exists in different forms with one or two copper binding sites (Lawton <i>et al.</i>, 2016) and CopD is a inner membrane protein that is presumably able to pump Cu(II) into the cell. We identified both genes in the genome of <i>P. brassicacearum</i> 3Re2-7 and transferred them into <i>E. coli</i> to increase the uptake of Cu(II) and accumulate intracellular copper.
-			<td>50</td>
+</article>
-		  </tr>
+<a name="con" id="con" class="shifted-anchor"></a>
-		  <tr>
+<h2>Conclusion</h2>
-			<td>Eve</td>
+<article>Our copper accumulation system works in two ways. We prevent the export of Cu(I) by knocking out the native <i>E. coli</i> copper export <i>cus</i> operon. In addition we identified genes involved in copper uptake, <i>hmtA</i>, <i>oprC</i>, <i>copC</i> and <i>copD</i>, and cloned them into <i> E. coli</i>. This combined system provides and represents an highly efficient and specific accumulation system for copper ions.
-			<td>Jackson</td>
+</article>
-			<td>94</td>
+<h2>The results of the accumulation experiments can be found <a href="https://2018.igem.org/Team:Bielefeld-CeBiTec/Accumulation_Results">here</a>.</h2>
-		  </tr>
-		  <tr>
-			<td>John</td>
-			<td>Doe</td>
-			<td>80</td>
-		  </tr>
-		</table>
--->
                     <hr style="width:60%"></hr>
-                   <h2>References</h2>
-                   <div class="reftext">
-                   Ladomersky, E., & Petris, M. J. (2015). Copper tolerance and virulence in bacteria. <i>Metallomics</i>, <i>7</i>(6), 957-964.<br/>
-                   Lewinson, O., Lee, A. T., & Rees, D. C. (2009). A P-type ATPase importer that discriminates between essential and toxic transition metals. <i>Proceedings of the National Academy of Sciences</i>, <i>106</i>(12), 4677-4682.<br>
-Pederick, V. G., Eijkelkamp, B. A., Begg, S. L., Ween, M. P., McAllister, L. J., Paton, J. C., & McDevitt, C. A. (2015). ZnuA and zinc homeostasis in Pseudomonas aeruginosa. <i>Scientific reports</i>, <i>5</i>, 13139.<br>
-Postle, K., & Good, R. F. (1983). DNA sequence of the Escherichia coli tonB gene. <i>Proceedings of the National Academy of Sciences</i>, <i>80</i>(17), 5235-5239.<br>
-Rensing, C., & Grass, G. (2003). Escherichia coli mechanisms of copper homeostasis in a changing environment. <i>FEMS microbiology reviews</i>, <i>27</i>(2-3), 197-213.<br>
-Solomon, E. I., Heppner, D. E., Johnston, E. M., Ginsbach, J. W., Cirera, J., Qayyum, M., ... & Tian, L. (2014). Copper active sites in biology. <i>Chemical reviews</i>, <i>114</i>(7), 3659-3853.<br>
-Xiao, M., Zhu, X., Fan, F., Xu, H., Tang, J., Qin, Y., ... & Zhang, X. (2017). Osmotolerance in Escherichia coli is improved by activation of copper efflux genes or supplementation with sulfur containing amino acids. <i>Applied and environmental microbiology</i>, AEM-03050.<br>
-Yoneyama, H., & Nakae, T. (1996). Protein C (OprC) of the outer membrane of Pseudomonas aeruginosa is a copper-regulated channel protein. <i>Microbiology</i>, <i>142</i>(8), 2137-2144.
-                   </div>
+<button onclick="myFunction()" class="refbtn"> References &#9662;</button>
+<div id="myDIV" class="reftext">
+                   <b>Arnesano, F., Banci, L., Bertini, I., & Thompsett, A. R. (2002).</b> Solution structure of CopC: a cupredoxin-like protein involved in copper homeostasis. <i>Structure</i>, <i>10</i>(10), 1337-1347.<br/>
-	    </div>
+<b>Bagai, I., Rensing, C., Blackburn, N. J., & McEvoy, M. M. (2008).</b> Direct metal transfer between periplasmic proteins identifies a bacterial copper chaperone. <i>Biochemistry</i>, <i>47</i>(44), 11408-11414.</br>
-      </div>
+<b>Cha, J. S., & Cooksey, D. A. (1993).</b> Copper hypersensitivity and uptake in Pseudomonas syringae containing cloned components of the copper resistance operon. <i>Applied and environmental microbiology</i>, <i>59</i>(5), 1671-1674.</br>
+<b>Delmar, J. A., Su, C. C., & Edward, W. Y. (2013).</b> Structural mechanisms of heavy-metal extrusion by the Cus efflux system. <i>Biometals</i>, <i>26</i>(4), 593-607.</br>
+<b>Franke, S., Grass, G., Rensing, C., & Nies, D. H. (2003).</b> Molecular analysis of the copper-transporting efflux system CusCFBA of Escherichia coli. <i>Journal of bacteriology</i>, <i>185</i>(13), 3804-3812.</br>
+<b>Gudipaty, S. A., Larsen, A. S., Rensing, C., & McEvoy, M. M. (2012).</b> Regulation of Cu (I)/Ag (I) efflux genes in Escherichia coli by the sensor kinase CusS. <i>FEMS microbiology letters</i>, <i>330</i>(1), 30-37.</br>
+<b>Ladomersky, E., & Petris, M. J. (2015).</b> Copper tolerance and virulence in bacteria. <i>Metallomics</i>, <i>7</i>(6), 957-964.</br>
+<b>Lawton, T. J., Kenney, G. E., Hurley, J. D., & Rosenzweig, A. C. (2016).</b> The CopC family: structural and bioinformatic insights into a diverse group of periplasmic copper binding proteins. <i>Biochemistry</i>, <i>55</i>(15), 2278-2290.</br>
+<b>Lewinson, O., Lee, A. T., & Rees, D. C. (2009).</b> A P-type ATPase importer that discriminates between essential and toxic transition metals. <i>Proceedings of the National Academy of Sciences</i>, <i>106</i>(12), 4677-4682.</br>
+<b>McDevitt, C. A. (2015).</b> ZnuA and zinc homeostasis in Pseudomonas aeruginosa. <i>Scientific reports</i>, <i>5</i>, 13139.</br>
+<b>Munson, G. P., Lam, D. L., Outten, F. W., & O'Halloran, T. V. (2000).</b> Identification of a copper-responsive two-component system on the chromosome of Escherichia coli K-12. <i>Journal of Bacteriology</i>, <i>182</i>(20), 5864-5871.</br>
+<b>Outten, F. W., Huffman, D. L., Hale, J. A., & O'Halloran, T. V. (2001).</b> The independent cue and cusSystems confer copper tolerance during aerobic and anaerobic growth inEscherichia coli. <i>Journal of Biological Chemistry</i>, <i>276</i>(33), 30670-30677.</br>
+<b>Pederick, V. G., Eijkelkamp, B. A., Begg, S. L., Ween, M. P., McAllister, L. J., Paton, J. C., & McDevitt, C. A. (2015).</b> ZnuA and zinc homeostasis in Pseudomonas aeruginosa. <i>Scientific reports</i>, <i>5</i>, 13139.</br>
+<b>Postle, K., & Good, R. F. (1983).</b> DNA sequence of the Escherichia coli tonB gene. <i>Proceedings of the National Academy of Sciences</i>, <i>80</i>(17), 5235-5239.</br>
+<b>Quintana, J., Novoa-Aponte, L., & Argüello, J. M. (2017).</b> Copper homeostasis networks in the bacterium Pseudomonas aeruginosa. <i>Journal of Biological Chemistry</i>, jbc-M117.</br>
+<b>Rensing, C., & Grass, G. (2003).</b> Escherichia coli mechanisms of copper homeostasis in a changing environment. <i>FEMS microbiology reviews</i>, <i>27</i>(2-3), 197-213.</br>
+<b>Solomon, E. I., Heppner, D. E., Johnston, E. M., Ginsbach, J. W., Cirera, J., Qayyum, M., ... & Tian, L. (2014).</b> Copper active sites in biology. <i>Chemical reviews</i>, <i>114</i>(7), 3659-3853.</br>
+<b>Tseng, T. T., Gratwick, K. S., Kollman, J., Park, D., Nies, D. H., Goffeau, A., & Saier Jr, M. H. (1999).</b> The RND permease superfamily: an ancient, ubiquitous and diverse family that includes human disease and development proteins. <i>Journal of molecular microbiology and biotechnology</i>, <i>1</i>(1), 107-125.</br>
+<b>Xiao, M., Zhu, X., Fan, F., Xu, H., Tang, J., Qin, Y., ... & Zhang, X. (2017).</b> Osmotolerance in Escherichia coli is improved by activation of copper efflux genes or supplementation with sulfur containing amino acids. <i>Applied and environmental microbiology</i>, AEM-03050.</br>
+<b>Yoneyama, H., & Nakae, T. (1996).</b> Protein C (OprC) of the outer membrane of Pseudomonas aeruginosa is a copper-regulated channel protein. <i>Microbiology</i>, <i>142</i>(8), 2137-2144.
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