Difference between revisions of "Team:Bielefeld-CeBiTec/Toxicity Theory"

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              <div class="sidenavi" id="side_bar">
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<div class="title">Accumulation Results</div>
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<li class="side_list">
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<a href="#gold">Gold</a>
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<a href="#silver">Silver</a>
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<a href="#copper">Copper</a>
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<a href="#iron">Iron</a>
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                        </li>
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                        <li class="side_list">
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<a href="#ros">Reactive oxygen species</a>
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<article>To test whether our accumulation system with the importers <i>oprC</i>, <i>hmtA</i>, <i>copC</i> and <i>copD</i> works as expected, we conducted experiments indicating Cu(II) ion uptake. We conducted growth experiments as due to its toxicity intracellular copper hinders cell growth and this would point to a working uptake system. We also conducted membrane permeability assays to show the location in the outer membrane and the channel nature of the proteins.</article>
  
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<h2>Toxicity assay</h2>
  
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      <div class="container">
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<div class="article">
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As intracellular copper triggers toxic effects on the cell (also see <a href="https://2018.igem.org/Team:Bielefeld-CeBiTec/Toxicity_Theory" target="_blank">Toxicity</a>), an increased uptake of Cu(II) ions should exacerbate cell growth. Therefore, we examined the growth of <i>E. coli</i> expressing <i>copC</i>, <i>copD</i>, <i>oprC</i>, <i>hmtA</i> and pSB1C3 as a control in lysogeny broth (LB) at different concentrations of CuSO<sub>4</sub> (0 mM, 1 mM, 2 mM, 3 mM, 4 mM, 8 mM) by measuring the optical density (OD) at a wavelength of 600 nm. The measurement was performed with the <a href="https://lifesciences.tecan.com/plate_readers/infinite_200_pro" target="_blank"> Infinite® 200 PRO</a> in a 24 wellplate with flat bottom (Greiner®). For expression the biobricks BBa_K525998 (T7 promoter with RBS) and a combination of BBa_I0500 (<i>pBAD/araC</i> promoter) and BBa_B0030 (RBS) were used each in combination with the basic parts BBa_K2638001 (<i>copC</i>), BBa_K2638002 (<i>copD</i>), BBa_K2638200 (<i>oprC</i>) and BBa_K2638000 (<i>hmtA</i>). The resulting parts are shown in table 1:
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</div>
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<table id="t01" class="centern" style="margin-top:30px; margin-bottom:30px;">
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<caption style="line-height:1.5; text.align:left;"><b>Table 1: </b>Parts used in toxicity assay (growth curves)</caption>
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  <tr>
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  <th>Biobrick number</th>
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        <th>Components</th>
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        <th>Function</th>
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  </tr>
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  <tr>
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        <th>BBa_K2638003</th>
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        <td>BBa_K525998, BBa_K2638001</td>
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  <td>T7, RBS, <i>copC</i></td>
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  </tr>
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      <tr>
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        <th>BBa_K2638004</th>
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        <td>BBa_K525998, BBa_K2638002</td>
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        <td>T7, RBS, <i>copD</i></td>
 +
  </tr>
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  <tr>
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        <th>BBa_K2638016</th>
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        <td>BBa_K525998, BBa_K2638000</td>
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        <td>T7, RBS, <i>hmtA</i></td>
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      </tr>
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      <tr>
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        <th>BBa_K2638201</th>
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        <td>BBa_K525998, BBa_K2638200</td>
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        <td>T7, RBS, <i>oprC</i></td>
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      </tr>
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      <tr>
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        <th>BBa_K2638005</th>
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        <td>BBa_I0500, BBa_B0030, BBa_K2638001</td>
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        <td>pBAD/araC, RBS, <i>copC</i></td>
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      </tr>
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      <tr>
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        <th>BBa_K2638006</th>
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        <td>BBa_I0500, BBa_B0030, BBa_K2638002</td>
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        <td>pBAD/araC, RBS, <i>copD</i></td>
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      </tr>
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      <tr>
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        <th>BBa_K2638204</th>
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        <td>BBa_I0500, BBa_B0030, BBa_K2638200</td>
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        <td>pBAD/araC, RBS, <i>oprC</i></td>
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      </tr>
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</table>
  
  
  
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          <div class="main_content">
 
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                  <div class="title">Toxicity</div>
 
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<article>
 
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Gold, silver, copper and iron ions are of interest due to their
 
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chemical properties. On the other hand some of the ions possess toxic properties
 
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for the cell (Ochoa-Herrera <i>et al.</i>, 2011; Shareena <i>et al.</i>, 2015;
 
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Zhao & Stevens, 2009). Therefore the toxicity of the different ions of the metals
 
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needs to be included into consideration when planning the uptake and processing
 
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of the ions as a substrate for nanoparticles. Not only is the toxicity of the
 
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dissolved ions of interest but also the potential toxic effect of nanoparticles
 
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on the cell.
 
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</article>
 
  
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<a name="gold" id="gold" class="shifted-anchor"></a>
 
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<h2>Gold</h2>
 
  
  
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<article>
 
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Gold nanoparticles (AuNPs) are popular in many scientific applications, e.g. the transfection of human cells (Chang <i>et al.</i>, 2008). However, there are several reports regarding its toxicity whose results differ greatly from each other (Brust <i>et al.</i>, 1995; Caruntu <i>et al.</i>, 2002; Zhang <i>et al.</i>, 2015). AuNPs in the form of nanorods could be identified as non-toxic, solely the coating agent was found to be toxic (Alkilany <i>et al.</i>, 2009). AuNPs in general do not possess toxic properties. Reported toxic effects of AuNPs result from poor purification of the AuNPs from Au(III) (Shareena Dasari, 2015).</br>
 
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Gold ions, such as Au(I) and Au(III), have toxic properties. Shareena Dasari <i>et al.</i> proved in 2015 that exposure to Au(I) and Au(III) inhibits the growth of nonpathogenic <i>Escherichia coli</i> as seen in figure 1. Furthermore they determined the half maximal inhibitory concentrations (IC<sub>50</sub>) for both ions in the examined buffers as seen in table 1, clearly indicating that gold ions possess toxic properties to <i>E. coli</i>. The toxic effect of gold ions is due to its ability to oxidative cleavage of peptide and protein disulfide bonds (Witkiewicz & Shaw, 1981).</br>
 
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Since gold ions are toxic to the NPs producing cell, the reduction to AuNPs is desirable, however the stress to the cell and its inhibitory effect have to be investigated.
 
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</article>
 
  
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<figure role="group">
 
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                      <img class="figure eighty" src="https://static.igem.org/mediawiki/2018/9/95/T--Bielefeld-CeBiTec--jr--gold_ions.png">
 
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                      <figcaption>
 
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                          <b>Figure 1:</b> Concentration dependent growth inhibition by Au(I) and Au(III) ions on multi-drug resistant <i>E. coli</i> (BAA-1161) according to Shareena Dasari (2015).
 
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                      </figcaption>
 
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                  </figure>
 
  
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<a name="silver" id="silver" class="shifted-anchor"></a>
 
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<h2>Silver</h2>
 
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<article>
 
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Silver nanoparticles (AgNPs) are known to possess toxic properties. The toxicity of AgNPs varies in response to the shape and size of the AgNPs. The toxicity increases significantly when the AgNPs are smaller than 10 nm (Ivask <i>et al.</i>, 2014). It has been demonstrated that AgNPs exhibit toxic effects in bacteria at environmentally relevant concentrations (Colman <i>et al.</i>, 2013). The growth inhibition is dependent on the concentration of the AgNPs. The minimal inhibitory concentration (MIC) for AgNPs in <i>E. coli</i> is considered to be between 3.3 and 6.6 nM (Kim <i>et al.</i>, 2007). The exposure to AgNPs results in an apoptosis-like response of the cell. To begin with, the membrane is depolarized by AgNPs, generating reactive oxygen species (ROS), such as hydroxyl peroxide, hydroxyl radicals and superoxide anions.These ROS cause oxidative stress and lead to the fragmentation of DNA. Simultaneously calcium accumulates in the cytoplasm of the cell. An increased concentration of calcium results in the inversion of phosphatidylserine in the membrane. All of these processes ultimately lead to the activation of bacterial caspase-like proteins and RecA (Bortner & Cidlowski, 2007; Yun & Lee, 2017). However there are considerations whether the AgNPs are toxic or whether the toxic effect is due to ions dissolving from the nanoparticles (Hwang <i>et al.</i>, 2008).</br>
 
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The toxic effects of silver salts have been known since antiquity. Even Hippocrates recognized its antimicrobial properties in water (Magner, 1992). The toxic effect of silver ions relies on the same chemical features as those of the AgNPs. When being exposed to silver ions, the bacterium experiences several forms of oxidative stress, mainly ROS which arise from Fenton chemistry. Ag(I) disrupts metabolic pathways that drive Fenton chemistry and lead to the overproduction of hydroxyl radicals and cell death (Morones-Ramirez <i>et al.</i>, 2013). At a concentration of 18.9 µM, all bacterial growth comes to an end (Zhao & Stevens, 1998).</br>
 
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Silver is toxic in both of its forms: as a nanoparticle and as dissolved ions. Therefore an approach to decrease the toxic effect on the cell is needed.
 
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</article>
 
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<figure role="group">
 
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                      <img class="figure eighty" src="https://static.igem.org/mediawiki/2018/2/29/T--Bielefeld-CeBiTec--JR-SilverrToxicity.jpeg">
 
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                      <figcaption>
 
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                          <b>Figure 2:</b> Toxic effects of silver nanoparticles and silver ions on the cell. Silver nanoparticles and silverer ions lead to a rising level of ROS which causes oxidative stress and damage to the DNA, proteins and the membrane.
 
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                      </figcaption>
 
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                  </figure>
 
  
  
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<!-- <span id="copper"></span> -->
 
  
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<a name="copper" id="copper" class="shifted-anchor"></a>
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<h2>Copper</h2>
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<article>
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Copper is known for its toxic and bactericidal properties and is often used as an antimicrobial agent in form of copper nanoparticles (CuNPs) (Cioffi <i>et al.</i>, 2005). Recent studies revealed that the inactivation and bactericidal activity works in a size-dependent manner. The smaller a CuNP is, the higher is its inhibitory potential to bacteria. Furthermore, the toxicity is probably elicited in a strain-specific manner in <i>E. coli</i> obtained from different sources (Alum <i>et al.</i>, 2018). CuNPs’ toxic properties appear to result from oxidative stress and protein damage, DNA damage and membrane damage. The generation of hydroxyl peroxide by CuNPs is assumed to be the reason for the toxicity of CuNPs. Li <i>et al.</i> could observe 2013 the release of copper and the production of Cu(I) ions.</br>
+
<!--
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Copper is considered to be essential in small amounts to both human and bacteria (Burgess <i>et al.</i>, 1999). It plays an important role in electron-transfer reactions (Kaplan & Lutsenko, 2009). Yet copper ions can induce cell death and exhibit toxic effects. This is due to copper ions interfering with the cell proteins or enzymes by chelating sulfhydryl groups and peroxidizing the lipids of the cell membrane (Yeager, 1991). A broad variety of microorganisms, such as <i>E. coli</i>, could be proven to be inhibited in growth or killed by the exposure to Cu(II) (Ochoa-Herrera <i>et al.</i>, 2011). Furthermore, copper ions can interact with oxygen by catalyzing Haber-Weiss and/or Fenton reactions. This results in the generation of reactive oxygen species which can ultimately damage biomolecules (Halliwell, 2007; Halliwell & Gutteridge, 2015).</br>
+
<figure role="group">
−
Copper is toxic in both of its forms: as a nanoparticle and as dissolved ions. Therefore an approach to decrease the toxic effect on the cell is needed.
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                       <img class="figure hundred" src="https://static.igem.org/mediawiki/2018/e/e3/T--Bielefeld-CeBiTec--recyclng_with_ferritin_vk.png">
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</article>
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                    <figure role="group">
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                       <img class="figure eighty" src="https://static.igem.org/mediawiki/2018/a/aa/T--Bielefeld-CeBiTec--JR-CopperToxicityNew.jpeg">
+
 
                       <figcaption>
 
                       <figcaption>
−
                           <b>Figure 3:</b> Toxic effects of copper nanoparticles and copper ions on the cell. Copper nanoparticles and copper ions lead to a rising level of ROS which causes oxidative stress and damage to the DNA, proteins and the membrane.  
+
                           <b>Figure 1:</b> Ferritin is suitable for metal recycling, since it can form e.g. iron, silver and gold nanoparticles in its cavity.  
 
                       </figcaption>
 
                       </figcaption>
 
                   </figure>
 
                   </figure>
 +
                 
 +
                 
 +
                 
 +
<table id="t01" class="centern" style="margin-top:30px; margin-bottom:30px;">
 +
<caption style="line-height:1.5; text.align:left;"><b>Table 1:</b>R and R<sup>2</sup> values of the tested constructs.</caption>
 +
  <tr>
 +
<th></th>
 +
<th>BBa_K2638112</th>
 +
                        <th>pSB1C3</th>
 +
                        <th>BBa_K2638114</th>
 +
                        <th>BBa_K2638118</th>
 +
                        <th>BBa_K2638110</th>
 +
  </tr>
 +
  <tr>
 +
<th>R value</th>
 +
<td>0.996</td>
 +
<td>0.952</td>
 +
<td>0.993</td>
 +
<td>0.987</td>
 +
<td>0.994</td>
 +
  </tr>
 +
  <tr>
 +
<th>R<sup>2</sup> value</th>
 +
<td>0.992</td>
 +
<td>0.907</td>
 +
<td>0.985</td>
 +
<td>0.973</td>
 +
<td>0.989</td>
 +
  </tr>
 +
</table>
 +
                 
 +
                 
 +
-->
  
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<a name="iron" id="iron" class="shifted-anchor"></a>
 
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<h2>Iron</h2>
 
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<article>
 
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Iron is crucial for certain biological functions and necessary for the cell in order to work properly. Especially when it comes to electron transfer reactions iron as a cofactor is critical for the formation of biomolecules (Zhang, 2014). However, free non-bound iron bears risks for the cell. Iron regularly appears as a ferrous (Fe(II)) or as a ferric ion (Fe(III)) and is able to accept an electron from neighboring molecules which leads to the generation of ROS and causes damage to cellular components. The generation of ROS occurs by Fenton and Haber-Weiss reactions (Kehrer, 2000; Winterbourn, 1995).</br>
 
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The desired iron nanoparticles shall be composed of Fe<sub>2</sub>O<sub>3</sub> which is one of the most abundant transition metal oxides and ubiquitous in the environment (Bi & Xu, 2012). Toxic effects by these nanoparticles arise from the formation of ROS by converting H<sub>2</sub>O<sub>2</sub> or O<sub>2</sub> (Huang <i>et al.</i>, 2015). Further studies performed by Wang <i>et al.</i> (2017) regarding the photocatalytic degradation of DNA who found that the majority of intracellular damage especially concerning the DNA arises from ROS.</br>
 
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</article>
 
  
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<a name="ros" id="ros" class="shifted-anchor"></a>
 
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<h2>Reactive oxygen species</h2>
 
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<article>
 
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As most problems from heavy metals arise from the generation of ROS, the efforts to improve the resistance to the toxic effects need to focus on the repair of the damage of biomolecules and the prevention of further damage.</br>
 
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Molecular oxygen itself cannot easily oxidize another molecule due to its spin restriction (Farr & Kogoma, 1991). However, certain cases allow oxidation of molecules by molecular oxygen, e.g. the presence of a paramagnetic center other than that of the molecular oxygen and good catalysts for one-electron reduction such as the transition metals copper and iron. The arising ROS are mostly the superoxide anion (O<sub>2</sub><sup>-</sup>), hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>), hydroxyl radicals (•OH) and singlet oxygen (<sup>1</sup>O<sub>2</sub>) (Halliwell, 1991).</br>
 
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O<sub>2</sub><sup>-</sup> attacks thiols and  ascorbate as well as iron sulfur clusters in proteins and thus disrupts the protein’s structure and leads to a loss of its function. Furthermore, it is able to reduce transition metals such as iron and copper, e.g. Cu(II) to Cu(I). It tends to form the highly reactive hydroperoxyl radical (•OOH) under acidic conditions. The degradation of the superoxide anion happens by spontaneous dismutation into hydrogen peroxide and molecular oxygen (Masoud <i>et al.</i>, 2015).</br>
 
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Hydrogen peroxide itself is reactive as well. The exact reaction mechanism in the cell is unknown due to its reaction speed. However, it is known that hydrogen peroxide reacts with reduced copper and/or iron ions to form hydroxyl radicals. As a weak oxidizing agent it attacks thiol groups and reduces glutathione (Liu <i>et al.</i>, 2015).</br>
 
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Hydroxyl radicals are highly reactive and react with almost every molecule. Since it is highly reactive, its reaction rate is limited by diffusion. The reactivity of hydroxyl radicals derives from its high standard electrode potential (+2.3 V). Therefore, it is able to oxidize almost any molecule other than ozone (Zhang <i>et al.</i>, 2018).</br>
 
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There are several origins for the different types of ROS: Superoxide anion is formed by autoxidation of distinct dehydrogenases and certain reductases, e.g. glutathione reductase. Non-enzymatic production is achieved by autoxidation of certain cellular components such as ubiquinols. Hydrogen peroxidase derives from several oxidases and the superoxide dismutase. UV radiation breaks it down into hydroxyl radicals (Mignolet-Spruyt <i>et al.</i>, 2016). Other mechanisms for the generation of hydroxyl radicals are the Fenton and/or Haber-Weiss reaction (Fagali <i>et al.</i>, 2015; Kehrer, 2000).</br>
 
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      <u>Haber-Weiss:</u> &emsp; &emsp; Fe<sup>3+</sup> + •O<sub>2</sub><sup>-</sup>    →    Fe<sup>2+</sup> + O<sub>2</sub></br>
 
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      <u>Fenton:</u>  &emsp; &emsp;    Fe<sup>2+</sup> + H<sub>2</sub>O<sub>2</sub>    →    Fe<sup>3+</sup> + OH<sup>-</sup> + •OH</br>
 
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In order to neutralize the ROS and repair the occured damage bacteria have different mechanisms they can rely on. Two types of superoxide dismutase (SOD) dismutate O<sub>2</sub><sup>-</sup> into H<sub>2</sub>O<sub>2</sub>: MnSOD and FeSOD (encoded by <i>sodA</i> and <i>sodB</i>) (Broxton & Culotta, 2016). Catalases (encoded by <i>katE</i> and <i>katG</i>) are able to disproportionate H<sub>2</sub>O<sub>2</sub> into the nontoxic components H<sub>2</sub>O and O<sub>2</sub> (Chaithawiwat <i>et al.</i>, 2016). Further detoxification is achieved by the alkyl hydroperoxide reductase Ahp (encoded by <i>ahpC</i> and <i>ahpF</i>) which scavenges various free organic hydroperoxides (Kamariah <i>et al.</i>, 2016). These genes are in <i>E. coli</i> under the control of various global regulators such as OxyR and SoxR. The regulators also promote the expression of genes involved in the repair of the damaged cell membranes, DNA and proteins and are activated by a conformational change induced by oxidative stress (Dubbs & Mongkolsuk, 2016; Seo <i>et al.</i>, 2015).</br>
 
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In order to increase the cell’s resistance to oxidative stress a sophisticated approach is needed. An overexpression of the oxidative stress dependent regulators SoxR and OxyR should improve the cell’s ability to cope with an elevated number of ROS. Various combinations of the defense mechanisms are tested in order to determine the ideal defense against oxidative stress in order to maximize the longevity in presence of heavy metals. Agents like phytochelatin which are rich in cysteine and/or histidine residues are meant to bind free metal ions to lower the potential for Fenton and/or Haber-Weiss reactions.
 
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<b>Molecular graphics and analyses performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311.</b> </br>
  
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  <b>Alkilany, A. M., Nagaria, P. K., Hexel, C. R., Shaw, T. J., Murphy, C. J., & Wyatt, M. D. (2009)</b>. Cellular uptake and cytotoxicity of gold nanorods: molecular origin of cytotoxicity and surface effects. Small, 5(6), 701-708.<br/>
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<b> Butts, C.A., Swift, J., Kang, S., Di Costanzo, L., Christianson, D.W., Saven, J.G., and Dmochowski, I.J. (2008).</b>. Directing Noble Metal Ion Chemistry within a Designed Ferritin Protein † , ‡. Biochemistry 47: 12729–12739.</br>
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                  <b>Alum, A., Alboloushi, A., & Abbaszadegan, M. (2018)</b>. Copper nanoparticles toxicity: Laboratory strains verses environmental bacterial isolates. Journal of Environmental Science and Health, Part A, 1-8.<br/>
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<b>Bi, D.Q., Xu, Y.M. (2012)</b>. Influence of iron oxide doping on the photocatalytic degradation of organic dye X3B over tungsten oxide. Acta Physico-Chimica Sinica, 28, 1777-1782.<br/>
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<b> Castro, L., Blázquez, M.L., Muñoz, J., González, F., and Ballester, A. (2014).</b>. Mechanism and Applications of Metal Nanoparticles Prepared by Bio-Mediated Process. Rev. Adv. Sci. Eng. 3.</br>
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<b>Bortner, C. D., Cidlowski, J. A. (2007)</b>. Cell shrinkage and monovalent cation fluxes: role in apoptosis. Archives of Biochemistry and Biophysics, 462(2), 176-188.<br/>
+
 
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<b>Broxton, C. N., & Culotta, V. C. (2016)</b>. SOD enzymes and microbial pathogens: surviving the oxidative storm of infection. PLoS pathogens, 12(1), e1005295.<br/>
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<b> Ensign, D., Young, M., and Douglas, T. (2004).</b>. Photocatalytic synthesis of copper colloids from CuII by the ferrihydrite core of ferritin. Inorg. Chem. 43: 3441–3446.</br>
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<b>Brust, M., Fink, J., Bethell, D., Schiffrin, D. J., & Kiely, C. (1995)</b>. Synthesis and reactions of functionalised gold nanoparticles. Journal of the Chemical Society, Chemical Communications, (16), 1655-1656.<br/>
+
 
−
<b>Burgess, J. E., Quarmby, J., & Stephenson, T. (1999)</b>. Role of micronutrients in activated sludge-based biotreatment of industrial effluents. Biotechnology advances, 17(1), 49-70.<br/>
+
<b> Goujon, M., McWilliam, H., Li, W., Valentin, F., Squizzato, S., Paern, J., and Lopez, R. (2010).</b>. A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res. 38: W695-699.</br>
−
<b>Caruntu D, Remond Y, Chou NH, Jun M-J, Caruntu G, He J, Goloverda G, O'Connor C, Kolesnichenko V (2002)</b>. Reactivity of 3d transition metal cations in diethylene glycol solutions. Synthesis of transition metal ferrites with the structure of discrete nanoparticles complexed with long-chain carboxylate anions. Inorganic chemistry, 41(23), 6137-6146.<br/>
+
 
−
<b>Chaithawiwat, K., Vangnai, A., McEvoy, J. M., Pruess, B., Krajangpan, S., & Khan, E. (2016)</b>. Role of oxidative stress in inactivation of Escherichia coli BW25113 by nanoscale zero-valent iron. Science of the Total Environment, 565, 857-862.<br/>
+
<b> Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., and Ferrin, T.E. (2004).</b>UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem 25: 1605–1612.</br>
−
<b>Cioffi, N., Torsi, L., Ditaranto, N., Tantillo, G., Ghibelli, L., Sabbatini, L., Bleve-Zacheo, T., D’Alessio, M., Zambonin, P.G., Traversa, E. (2005)</b>. Copper nanoparticle/polymer composites with antifungal and bacteriostatic properties. Chemistry of Materials, 17(21), 5255-5262.<br/>
+
 
−
<b>Chang, M. L., Chen, J. C., Yeh, C. T., Chang, M. Y., Liang, C. K., Chiu, C. T., Lin D.Y., Liaw, Y. F. (2008)</b>. Gene gun bombardment with DNA-coated gold particles is a potential alternative to hydrodynamics-based transfection for delivering genes into superficial hepatocytes. Human gene therapy, 19(4), 391-395.<br/>
+
<b> Sievers, F., Wilm, A., Dineen, D., Gibson, T.J., Karplus, K., Li, W., Lopez, R., McWilliam, H., Remmert, M., Söding, J., Thompson, J.D., and Higgins, D.G. (2011). </b>Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7: 539.</br>
−
<b>Colman, B. P., Arnaout, C. L., Anciaux, S., Gunsch, C. K., Hochella Jr, M. F., Kim, B., Lowry, G.V., McGill, B.M., Reinsch, B.C., Richardson, C.J., Unrine, J. M., Wright, J.P., Yin, L., Bernhardt, E.S. (2013)</b>. Low concentrations of silver nanoparticles in biosolids cause adverse ecosystem responses under realistic field scenario. PLoS One, 8(2), e57189.<br/>
+
 
−
<b>Dubbs, J. M., & Mongkolsuk, S. (2016)</b>. Peroxide‐Sensing Transcriptional Regulators in Bacteria. Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria, 587-602.<br/>
+
<b> Ummartyotin, S., Bunnak, N., Juntaro, J., Sain, M., and Manuspiya, H. (2012). </b>. DSynthesis of colloidal silver nanoparticles for printed electronics. /data/revues/16310748/v15i6/S1631074812000549/.</br>
−
<b>Eid, R., Arab, N.T.T., Greenwood, M.T. (2017)</b>. Iron mediated toxicity and programmed cell death: A review and a re-examination of existing paradigms. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1864(2), 399-430.<br/>
+
 
−
<b>Fagali, N. S., Grillo, C. A., Puntarulo, S., & Lorenzo, M. A. F. (2015)</b>. Biodegradation of metallic biomaterials: its relation with the generation of reactive oxygen species. REACTIVE OXYGEN SPECIES, LIPID PEROXIDATION AND PROTEIN OXIDATION, 127.<br/>
+
<b> Wang, L., Hu, C., and Shao, L. (2017a).</b>. The antimicrobial activity of nanoparticles: present situation and prospects for the future. Int. J. Nanomedicine 12: 1227–1249.</br>
−
<b>Farr, S. B., Kogoma, T. (1991)</b>. Oxidative Stress Responses in Escherichia coli and Salmonella typhimurium. Microbiological Reviews, 55(4), 561-585.<br/>
+
 
−
<b>Halliwell, B. (1991)</b>. Reactive oxygen species in living systems: Source, biochemistry, and role in human disease. The American Journal of Medicine, 91(3), S14-S22.<br/>
+
<b> Wang, Z., Gao, H., Zhang, Y., Liu, G., Niu, G., and Chen, X. (2017b).</b>. Functional ferritin nanoparticles for biomedical applications. Front. Chem. Sci. Eng. 11: 633–646.</br>
−
<b>Halliwell, B. (2007)</b>. Biochemistry of oxidative stress. Biochem Soc Trans, 35(5), 1147-1150.<br/>
+
             
−
<b>Halliwell, B., & Gutteridge, J. M. (2015)</b>. Free radicals in biology and medicine. Oxford University Press, USA.<br/>
+
                  </div>
−
<b>Holleman, A. F.; Wiberg, N. (2001)</b>. Inorganic Chemistry. San Diego: Academic Press.<br/>
+
 
−
<b>Huang, Q., Cao, M.H., Ai, Z.H., Zhang, L.Z. (2015)</b>. Reactive oxygen species dependent degradation pathway of 4-chlorophenol with Fe@Fe2O3 core-shell nanowires. Appl Catal B-Environ, 162, 319-326.<br/>
+
</div>
−
<b>Hwang, E. T., Lee, J. H., Chae, Y. J., Kim, Y. S., Kim, B. C., Sang, B. I., & Gu, M. B. (2008)</b>. Analysis of the toxic mode of action of silver nanoparticles using stress‐specific bioluminescent bacteria. Small, 4(6), 746-750.<br/>
+
−
<b>Ivask, A., ElBadawy, A., Kaweeteerawat, C., Boren, D., Fischer, H., Ji, Z., Chang, C.H., Liu, R., Tolaymat, T., Telesca, D., Zink, J. I., Cohen, Y., Holden, P.A., Godwin, H.A., (2013)</b>. Toxicity mechanisms in Escherichia coli vary for silver nanoparticles and differ from ionic silver. Acs Nano, 8(1), 374-386.<br/>
+
−
<b>Kamariah, N., Nartey, W., Eisenhaber, B., Eisenhaber, F., & Grüber, G. (2016)</b>. Low resolution solution structure of an enzymatic active AhpC10: AhpF2 ensemble of the Escherichia coli Alkyl hydroperoxide Reductase. Journal of structural biology, 193(1), 13-22.<br/>
+
−
<b>Kaplan, J. H., & Lutsenko, S. (2009)</b>. Copper transport in mammalian cells: special care for a metal with special needs. Journal of Biological Chemistry, 284(38), 25461-25465.<br/>
+
−
<b>Kehrer, J.P. (2000)</b>. The Haber-Weiss reaction and mechanisms of toxicity. Toxicology, 149, 43-50.<br/>
+
−
<b>Kim, J.S., Kuk, E., Yu, K.N., Kim, J.H., Park, S.J., Lee, H.J., Kim, S.H., Park, Y.K., Park, Y.H., Hwang, C.Y., Kim, Y.K., Lee, Y.S., Jeong, D.H., Cho, M.H., (2007)</b>. Antimicrobial effects of silver nanoparticles. Nanomedicine: Nanotechnology, Biology, and Medicine, 3, 95-101.<br/>
+
−
<b>Letelier, M. E., Sánchez-Jofré, S., Peredo-Silva, L., Cortés-Troncoso, J., & Aracena-Parks, P. (2010)</b>. Mechanisms underlying iron and copper ions toxicity in biological systems: Pro-oxidant activity and protein-binding effects. Chemico-biological interactions, 188(1), 220-227.<br/>
+
−
<b>Li, F., Lei, C., Shen, Q., Li, L., Wang, M., Guo, M., Huang, Y., Nie, Z., Yao, S. (2013)</b>. Analysis of copper nanoparticles toxicity based on a stress-responsive bacterial biosensor array. Nanoscale, 5(2), 653-662.<br/>
+
−
<b>Liu, L., Wu, D., Zhao, B., Han, X., Wu, J., Hou, H., & Fan, Y. (2015)</b>. Copper (II) coordination polymers: tunable structures and a different activation effect of hydrogen peroxide for the degradation of methyl orange under visible light irradiation. Dalton Transactions, 44(3), 1406-1411.<br/>
+
−
<b>Magner, L.N. (1992)</b>. In: Hippocrates and the Hippocratic Tradition. A History of Medicine. Duffy J, editor. Marcel Dekker, Inc; NYC: 1992. p. 393.<br/>
+
−
<b>Masoud, R., Bizouarn, T., Trepout, S., Wien, F., Baciou, L., Marco, S., & Levin, C. H. (2015)</b>. Titanium dioxide nanoparticles increase superoxide anion production by acting on NADPH oxidase. PloS one, 10(12), e0144829.<br/>
+
−
<b>Mignolet-Spruyt, L., Xu, E., Idänheimo, N., Hoeberichts, F. A., Mühlenbock, P., Brosché, M. & Kangasjärvi, J. (2016)</b>. Spreading the news: subcellular and organellar reactive oxygen species production and signalling. Journal of Experimental Botany, 67(13), 3831-3844.<br/>
+
−
<b>Morones-Ramirez, J. R., Winkler, J. A., Spina, C. S., & Collins, J. J. (2013)</b>. Silver enhances antibiotic activity against gram-negative bacteria. Science translational medicine, 5(190), 190ra81-190ra81.<br/>
+
−
<b>Ochoa-Herrera, V., León, G., Banihani, Q., Field, J. A., Sierra-Alvarez, R. (2011)</b>. Toxicity of copper (II) ions to microorganisms in biological wastewater treatment systems. Science of the total environment, 412, 380-385.<br/>
+
−
<b>Seo, S. W., Kim, D., Szubin, R., & Palsson, B. O. (2015)</b>. Genome-wide reconstruction of OxyR and SoxRS transcriptional regulatory networks under oxidative stress in Escherichia coli K-12 MG1655. Cell Reports, 12(8), 1289-1299.<br/>
+
−
<b>Shareena Dasari, TP, Zhang, Y, Yu, H (2015)</b>. Antibacterial Activity and Cytotoxicity of Gold (I) and (III) Ions and Gold Nanoparticles. Biochem Pharmacol (Los Angel) 4(6), 199.<br/>
+
−
<b>Wang, X., Gu, Y., Johnson, D., Chen, C., Huang, Y. (2017)</b>. The toxicity and DNA-damage mechanism of α-Fe2O3 nanoparticles. Medicinal chemistry Research, 26(2), 384-389.<br/>
+
−
<b>Winterbourn, C.C. (1995)</b>. Toxicity of iron and hydrogen peroxide: the Fenton reaction. Toxicol. Lett., 82-83, 969-974.<br/>
+
−
<b>Witkiewicz, P. L., & Shaw, C. F. (1981)</b>. Oxidative cleavage of peptide and protein disulphide bonds by gold (III): a mechanism for gold toxicity. Journal of the Chemical Society, Chemical Communications, (21), 1111-1114.<br/>
+
−
<b>Yaeger C.C. (1991)</b>. Copper and zinc preservatives. In S.S. Block (ed.), Disinfection, sterilization, and preservation. 4th edn, Lea & Fiebiger Press, Philadelphia, USA, 358-361.<br/>
+
−
<b>Yun, J., Lee, D.G. (2017)</b>. Silver Nanoparticles: A Novel Antimicrobial Agent. Antimicrobial Nanoarchitectonics, 139-166.<br/>
+
−
<b>Zhang, C. (2014)</b>. Essential functions of iron-requiring proteins in DNA replication, repair and cell cycle control. Protein Cell, 5, 750-760.<br/>
+
−
<b>Zhang, Y., Newton, B., Lewis, E., Fu, P. P., Kafoury, R., Ray, P. C., & Yu, H. (2015)</b>. Cytotoxicity of organic surface coating agents used for nanoparticles synthesis and stability. Toxicology in Vitro, 29(4), 762-768.<br/>
+
−
<b>Zhang, H., Jiang, X., Cao, G., Zhang, X., Croley, T. R., Wu, X., & Yin, J. J. (2018)</b>. Effects of noble metal nanoparticles on the hydroxyl radical scavenging ability of dietary antioxidants. Journal of Environmental Science and Health, Part C, 36(2), 84-97.
+
−
<br/>
+
−
<b>Zhao, G, Stevens, SE Jr. (1998)</b>. Multiple parameters for the comprehensive evaluation of the susceptibility of Escherichia coli to the silver ion. BioMetals 11(1), 27-32.<br/>
+
−
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Revision as of 18:04, 17 October 2018

Accumulation Results
To test whether our accumulation system with the importers oprC, hmtA, copC and copD works as expected, we conducted experiments indicating Cu(II) ion uptake. We conducted growth experiments as due to its toxicity intracellular copper hinders cell growth and this would point to a working uptake system. We also conducted membrane permeability assays to show the location in the outer membrane and the channel nature of the proteins.

Toxicity assay

As intracellular copper triggers toxic effects on the cell (also see Toxicity), an increased uptake of Cu(II) ions should exacerbate cell growth. Therefore, we examined the growth of E. coli expressing copC, copD, oprC, hmtA and pSB1C3 as a control in lysogeny broth (LB) at different concentrations of CuSO4 (0 mM, 1 mM, 2 mM, 3 mM, 4 mM, 8 mM) by measuring the optical density (OD) at a wavelength of 600 nm. The measurement was performed with the Infinite® 200 PRO in a 24 wellplate with flat bottom (Greiner®). For expression the biobricks BBa_K525998 (T7 promoter with RBS) and a combination of BBa_I0500 (pBAD/araC promoter) and BBa_B0030 (RBS) were used each in combination with the basic parts BBa_K2638001 (copC), BBa_K2638002 (copD), BBa_K2638200 (oprC) and BBa_K2638000 (hmtA). The resulting parts are shown in table 1:
Table 1: Parts used in toxicity assay (growth curves)
Biobrick number Components Function
BBa_K2638003 BBa_K525998, BBa_K2638001 T7, RBS, copC
BBa_K2638004 BBa_K525998, BBa_K2638002 T7, RBS, copD
BBa_K2638016 BBa_K525998, BBa_K2638000 T7, RBS, hmtA
BBa_K2638201 BBa_K525998, BBa_K2638200 T7, RBS, oprC
BBa_K2638005 BBa_I0500, BBa_B0030, BBa_K2638001 pBAD/araC, RBS, copC
BBa_K2638006 BBa_I0500, BBa_B0030, BBa_K2638002 pBAD/araC, RBS, copD
BBa_K2638204 BBa_I0500, BBa_B0030, BBa_K2638200 pBAD/araC, RBS, oprC
Molecular graphics and analyses performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311.
Butts, C.A., Swift, J., Kang, S., Di Costanzo, L., Christianson, D.W., Saven, J.G., and Dmochowski, I.J. (2008).. Directing Noble Metal Ion Chemistry within a Designed Ferritin Protein † , ‡. Biochemistry 47: 12729–12739.
Castro, L., Blázquez, M.L., Muñoz, J., González, F., and Ballester, A. (2014).. Mechanism and Applications of Metal Nanoparticles Prepared by Bio-Mediated Process. Rev. Adv. Sci. Eng. 3.
Ensign, D., Young, M., and Douglas, T. (2004).. Photocatalytic synthesis of copper colloids from CuII by the ferrihydrite core of ferritin. Inorg. Chem. 43: 3441–3446.
Goujon, M., McWilliam, H., Li, W., Valentin, F., Squizzato, S., Paern, J., and Lopez, R. (2010).. A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res. 38: W695-699.
Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., and Ferrin, T.E. (2004).UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem 25: 1605–1612.
Sievers, F., Wilm, A., Dineen, D., Gibson, T.J., Karplus, K., Li, W., Lopez, R., McWilliam, H., Remmert, M., Söding, J., Thompson, J.D., and Higgins, D.G. (2011). Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7: 539.
Ummartyotin, S., Bunnak, N., Juntaro, J., Sain, M., and Manuspiya, H. (2012). . DSynthesis of colloidal silver nanoparticles for printed electronics. /data/revues/16310748/v15i6/S1631074812000549/.
Wang, L., Hu, C., and Shao, L. (2017a).. The antimicrobial activity of nanoparticles: present situation and prospects for the future. Int. J. Nanomedicine 12: 1227–1249.
Wang, Z., Gao, H., Zhang, Y., Liu, G., Niu, G., and Chen, X. (2017b).. Functional ferritin nanoparticles for biomedical applications. Front. Chem. Sci. Eng. 11: 633–646.