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

Revision as of 17:15, 17 October 2018

Toxicity Results

Abstract

In our experiments to improve the tolerance of Escherichia coli DH5alpha to heavy metals, we created a collection of several composite parts designed to fight oxidative stress. These parts mainly convey the ability to dismutate the superoxide anion and its secondary products like hydrogen peroxide into nontoxic forms. We were able to demonstrate that cells carrying our construct BBa_K2638118 or BBa_K2638112 does not lead to anare not subject to increased growth rate but to a significantly higher viability when the cells are exposed to elevated concentrations of heavy metals, namely cupric sulfate.

Evaluation

Heavy metal exposure poses many risks and dangers to living organisms and the environment. Certain heavy metal ions such as copper can interact with enzymes and lower their activity as well as their specificity. Reactive oxygen species (ROS) arise from processes such as Fenton chemistry and Haber-Weiss reactions. Therefore, a sophisticated approach to lower the toxic effects of heavy metals on the cell is desired. We evaluated several approaches of applying anti-oxidants against the generation of ROS. In our project, we set a focus on the accumulation of copper ions. Furthermore, using cupric salts is cheaper than gold ions and easier to solve than ferric salts. Its toxicity is lower than that of silver ions. Hence there is a broader spectrum in which anti-toxic measures can be explored. Therefore, we tested our approaches on anti-oxidant measures in different concentrations of cupric salts. Subject to our research were the five following composite parts: BBa_K2638109, BBa_K2638112, BBa_K2638114, BBa_K2638110 and BBa_K2638118.

**Figure 1:** Plotting the OD₆₀₀ of *E. coli* DH5alpha carrying an empty pSB1C3 plasmid versus time with varying copper concentrations. Experiments were performed in a total volume of 1 mL in a 24-well plate at 37 °C and 350 rpm (n=3).

Escherichia coli DH5alpha carrying an empty pSB1C3 vector was grown in LB medium containing different concentrations of cupric salt over a period of 13 hours. With no additional cupric salt added the cells reached an optical density of 0.848 and entered the stationary phase after 6.5 hours. Small additions of cupric salt to the medium appeared to have a benevolent effect on the growth: concentrations of 1 mM and 2 mM CuSO₄ resulted in a higher optical density of around 0.950. Growth started to deteriorate at a concentration of 4 mM CuSO₄. The maximal growth rate µmax dropped to 7.54E-3 min^-1 in comparison to the growth in absence of cupric salt (12.06E-3 min^-1) meaning it is only 62.52 % of the original µmax. Growth decreased even further with rising copper ion concentrations to 0.28E-3 min^-1 and 0.36E-3 min^-1. For the examination of antitoxic measures, three concentrations of cupric salts were chosen: 0 mM, 4 mM and 8 mM. 0 mM was used as a control. 4 mM CuSO₄ was chosen since growth could still be observed, even though growth was not as good as at lower concentrations of cupric salt. At 8 mM CuSO₄, no significant growth was observed. Therefore, it was interesting to see whether more growth occurs or whether a higher viability can be achieved by applying our approaches for antitoxic measures.

**Figure 2:** Plotting the OD₆₀₀ of *E. coli* DH5alpha each carrying different plasmid versus cultivation time in a volume of 1 mL LB medium containing 0 mM CuSO₄. Experiments were performed in a 24-well plate at 37 °C and 350 rpm (n=3).

The growth of strains carrying different constructs in absence of cupric salt was examined. No significant difference between the highest OD₆₀₀ values of the cells could be observed. However, cells carrying plasmids under the control of the pBAD promoter showed a different growth pattern since they can rely on another carbon source once glucose is metabolized. This leads to two consecutive exponential phases resulting in similar OD₆₀₀ values compared to those only relying on glucose as a carbon source. Other than that, no significant changes in growth behavior were observed.

**Figure 3:** Plotting the OD₆₀₀ of *E. coli* DH5alpha each carrying different plasmids versus time in a volume of 1 mL LB medium containing 4 mM CuSO₄. Experiments were performed in a 24-well plate at 37 °C and 350 rpm (n=3).

No mutant carrying any of the BioBricks exhibits an advantage regarding the growth rate or the final OD₆₀₀ value. While BBa_K2638110 and BBa_K2638114 exhibit the same growth pattern as the mutant carrying an empty pSB1C3 vector, BBa_K2638112 and BBa_K2638109 showed significant lower growth rates and did not reach an OD₆₀₀ value as high as the other mutants. The construct BBa_K2638118 achieved an OD₆₀₀ value of 0.79.

**Figure 4:** Plotting the OD of *E. coli* DH5alpha each carrying different plasmid versus time in a volume of 1 mL LB medium containing 8 mM CuSO₄. Experiments were performed in a 24-well plate at 37 °C and 350 rpm (n=3).

Every mutant showed visible growth only during the first two hours at best. Afterwards, there was no significant increase in the OD₆₀₀ value. The highest OD₆₀₀ values were achieved by the construct BBa_K2638112, the lowest OD₆₀₀ values by the construct BBa_K2638109.
However, since no growth improving effect using our initial approaches against toxicity could be observed, we decided to conduct an experiment based on the viability of the cells at the measured points of time at the beginning of the experiment and after three, six and 9 hours of cultivation.
We grew E. coli containing the constructs BBa_K2638112, BBa_K2638110, BBa_K2638110, BBa_K2638118 and pSB1C3 in 10 ml LB medium supplemented with 30 ng/ml chloramphenicol for 9 hours and either 0 mM, 4 mM or 8 mM CuSO₄. Every three hours, a sample was taken and 50 µl were plated in the following dilutions: 10^-4, 10^-5 and 10^-6. Additionally, whenever a sample was taken, the optical density at 600 nm was measured.
In order to validate our results, we correlated the optical density and the determined CFU (Colony Forming Units).

**Figure 5:** Plotting of the optical density at 600 nm of cells grown in LB medium containing 30 ng/ml chloramphenicol and no cupric salt at 37 °C against the amount of colony forming units (CFU) in ml^-1, as well as linear fits (n=3).

The OD₆₀₀ clearly correlates with the amount of colony forming units (CFU) when no further cupric sulfate was added to the medium. The Pearson correlation coefficient R of the different constructs can be found in the table below, indicating a significant positive correlation between the two values. The adjoined coefficients of determination R² implicate that at least 90 % of all changes in the optical density result in a change in the amount of colony forming units. This value increases even further regarding the different implemented constructs to at least 97.3 %.

**Table 1:**R and R² values of the tested constructs.
	BBa_K2638112	pSB1C3	BBa_K2638114	BBa_K2638118	BBa_K2638110
R value	0.996	0.952	0.993	0.987	0.994
R² value		0.992	0.907	0.985	0.973	0.989

The same measurements were performed for cells at cupric sulfate concentrations of 4 mM and 8 mM. The linear fits for the constructs grown in both supplemented media are shown in figures 6 and 7.

**Figure 6:** Plotting of the optical density at 600 nm of cells grown in LB medium containing 30 ng/ml chloramphenicol and 4 mM CuSO₄ at 37 °C against the amount of colony forming units (CFU) in ml^-1, as well as linear fits (n=3).

A positive correlation was indicated by determining the Pearson correlation coefficient and the coefficient for determination for the different constructs. However, according to its coefficient of determination, in the case of BBa_K2638110 only 83.7 % of all changes of the optical density could be referred to the changes in the amount of colony forming units,. according to its coefficient of determination. In general, a positive correlation between the optical density and the CFU could be clearly determined.

**Table 2:**R and R² values of the tested constructs.
	BBa_K2638112	pSB1C3	BBa_K2638114	BBa_K2638118	BBa_K2638110
R value	0.953	0.998	0.968	0.972	0.915
R² value		0.908	0.997	0.937	0.945	0.837

In contrast to those cells grown in the absence of cupric sulfate and in the presence of 4 mM cupric sulfate, no positive correlation between the optical density and the CFU could be observed. Instead, the cells grown in LB medium with 8 mM cupric sulfate were proven to show a negative correlation. This is clearly visible in figure 7 (see below).

**Figure 7:** Plotting of the optical density at 600 nm of cells grown in LB medium containing 30 ng/ml chloramphenicol and 8 mM CuSO₄ at 37 °C against the amount of colony forming units (CFU) in ml^-1, as well as linear fits (n=3).

The negative correlation was revealed by Pearson correlation coefficient R and coefficient of determination R².

**Table 2:**R and R² values of the tested constructs.
	BBa_K2638112	pSB1C3	BBa_K2638114	BBa_K2638118	BBa_K2638110
R value	-0.576	-0.955	-0.817	-0.952	-0.924
R² value		0.332	0.913	0.668	0.906	0.853

R2 was less than half as big as the R2 values of the other constructs and of all constructs at different concentrations of cupric sulfate. The negative correlation was likely due to cells dying at higher copper concentrations but not being degraded immediately so that the OD600 value suggests a higher cell density than viability. Therefore, the correlation of every construct shifts to the negative area. Furthermore, the highest R and R2 values have been determined for the mutant carrying the empty pSB1C3 vector.

**Figure 8:** Bar chart of the CFUs of the tested constructs in LB medium supplemented with either 0 mM or 4 mM cupric sulfate at 37 °C (n=3).

No significant difference in growth or viability in comparison to the empty pSB1C3 vector was observed for any tested construct at a concentration of either 0 mM or 4 mM cupric sulfate. According to the previously recorded growth trends, the growth rate decreased visibly with a rising concentration of copper ions. However, since no effect significantly improving the amount of colony forming units compared to the wildtype could be determined for these concentrations, the CFU at 8 mM cupric sulfate was evaluated.

**Figure 9:** Bar chart of the CFUs of the tested constructs in LB medium supplemented with 8 mM cupric sulfate at 37 °C (n=3), as well as the assigned significance levels to explain which constructs differ significantly from each other.

The number of colony forming units at the beginning of the measurement, after 0 hours, already shows indicates a great difference. Furthermore, the CFU in the course of time reveals differences for certain constructs in comparison to the wildtype. BBa_K2638112 and BBa_K2638118 appear to be more viable after 3, 6 and 9 hours of cultivation in 8 mM cupric sulfate containing medium. However, since no growth appeared to occur, the evaluation of this data concentrates on the preservation of the existing biomass rather than to improve its growth capabilities. After all, the cells are meant to be incubated and not cultivated in our remediation process of filtering water.
To examine the collected data regarding the influence of time and tested constructs, we conducted a two-factor ANOVA (analysis of variance) with repeated measurements for both factors. The aim is to discard the null hypothesis stating that there is no significant difference between the temporal developments of the tested constructs. Therefore, we conducted Mauchly’s Test of Sphericity to determine the samples sphericity. The test suggested highly with high significance to accept the assumption of sphericity with a significance level of .000 meaning that a correction of values by Greenhouse-Geisser was needed. The test of Within-Subject Effects resulted in the conclusion that the applied construct as well as the combination of the temporal development and the construct has a significant influence on the amount of colony forming units. In case of the construct, the influence was determined to be even highly significant. The constructs, the temporal development and the combination of those factors were examined, and each assigned to a significance level.
In conclusion, we were able to develop two composite parts which increase a cell’s tolerance for elevated concentrations of copper. This ameliorated tolerance could be verified for concentrations up to 8 mM of CuSO4 for a duration of up to 9 hours. This is a great first approach to realize our project and enable our cells to survive longer in the mining drainage. As our composite can also be used by iGEM teams in the future, we deem these results to be very useful to the community. Further applications could also be in the remediation of contaminated soils and seas, as well as industrial applications and processes in a heavy metal rich environment.

@@ Line 58: / Line 58: @@
 <div class="title">Toxicity Results</div>
-<h2>Original Part: BBa_K1189019</h2>
+<h2>Abstract</h2>
 <article>
-The Calgary 2013 iGEM team used the human ferritin wild type (<a href="http://parts.igem.org/Part:BBa_K1189019">BBa_K1189019</a>) as reporter protein on a test strip. They expressed the human ferritin heavy and light chain heterologous using Escherichis coli.  In the cells, the ferritin produced its characteristic iron core. With the help of fenton chemistry they were able to dye the iron core by producing the prussian blue iron complex. Beside the function as reporter, the team mentioned the capability of ferritin to produce nanoparticles with other metal ions.
+In our experiments to improve the tolerance of Escherichia coli DH5alpha to heavy metals, we created a collection of several composite parts designed to fight oxidative stress. These parts mainly convey the ability to dismutate the superoxide anion and its secondary products like hydrogen peroxide into nontoxic forms. We were able to demonstrate that cells carrying our construct BBa_K2638118 or BBa_K2638112 does not lead to anare not subject to increased growth rate but to a significantly higher viability when the cells are exposed to elevated concentrations of heavy metals, namely cupric sulfate.
 </article>
-  <figure role="group">
+<h2>Evaluation</h2>
-                       <img class="figure hundred" src="https://static.igem.org/mediawiki/2018/e/e3/T--Bielefeld-CeBiTec--recyclng_with_ferritin_vk.png">
+<div class="article">
+Heavy metal exposure poses many risks and dangers to living organisms  and the environment. Certain heavy metal ions such as copper can interact with enzymes and lower their activity as well as their specificity. Reactive oxygen species (ROS) arise from processes such as Fenton chemistry and Haber-Weiss reactions. Therefore, a sophisticated approach to lower the toxic effects of heavy metals on the cell is desired. We evaluated several approaches of applying anti-oxidants against the generation of ROS.
+In our project, we set a focus on the accumulation of copper ions. Furthermore, using cupric salts is cheaper than gold ions and easier to solve than ferric salts. Its toxicity is lower than that of silver ions. Hence there is a broader spectrum in which anti-toxic measures can be explored. Therefore, we tested our approaches on anti-oxidant measures in different concentrations of cupric salts.
+Subject to our research were the five following composite parts: <a href="http://parts.igem.org/Part:BBa_K2638109">BBa_K2638109</a>, <a href="http://parts.igem.org/Part:BBa_K2638112">BBa_K2638112</a>, <a href="http://parts.igem.org/Part:BBa_K2638114">BBa_K2638114</a>, <a href="http://parts.igem.org/Part:BBa_K2638110">BBa_K2638110</a> and <a href="http://parts.igem.org/Part:BBa_K2638118">BBa_K2638118</a>.
+</div>
+<figure role="group">
+                       <img class="figure hundred" src="https://static.igem.org/mediawiki/parts/1/13/T--Bielefeld-CeBiTec--jr--controlcopper.jpeg">
                        <figcaption>
-                            <b>Figure 1:</b> Ferritin is suitable for metal recycling, since it can form e.g. iron, silver and gold nanoparticles in its cavity.
+                            <b>Figure 1:</b> Plotting the OD<sub>600</sub> of <i>E. coli</i> DH5alpha carrying an empty pSB1C3 plasmid versus time with varying copper concentrations. Experiments were performed in a total volume of 1 mL in a 24-well plate at 37 °C and 350 rpm (n=3).
                        </figcaption>
                     </figure>
+<article>
+<i>Escherichia coli</i> DH5alpha carrying an empty pSB1C3 vector was grown in LB medium containing different concentrations of cupric salt over a period of 13 hours. With no additional cupric salt added the cells reached an optical density of 0.848 and entered the stationary phase after 6.5 hours. Small additions of cupric salt to the medium appeared to have a benevolent effect on the growth: concentrations of 1 mM and 2 mM CuSO<sub>4</sub> resulted in a higher optical density of around 0.950. Growth started to deteriorate at a concentration of 4 mM CuSO<sub>4</sub>. The maximal growth rate µmax dropped to 7.54E-3 min<sup>-1</sup> in comparison to the growth in absence of cupric salt (12.06E-3 min<sup>-1</sup>) meaning it is only 62.52 % of the original µmax. Growth decreased even further with rising copper ion concentrations to 0.28E-3 min<sup>-1</sup> and 0.36E-3 min<sup>-1</sup>. For the examination of antitoxic measures, three concentrations of cupric salts were chosen: 0 mM, 4 mM and 8 mM. 0 mM was used as a control. 4 mM CuSO<sub>4</sub> was chosen since growth could still be observed, even though growth was not as good as  at lower concentrations of cupric salt. At 8 mM CuSO<sub>4</sub>, no significant growth was observed. Therefore, it was interesting to see whether more growth occurs or whether a higher viability can be achieved by applying our approaches for antitoxic measures.
+</article>
-                   <hr style="width:60%"></hr>
+<figure role="group">
+                      <img class="figure hundred" src="https://static.igem.org/mediawiki/parts/b/b5/T--Bielefeld-CeBiTec--jr--0_mM_CuSO4.png">
+                      <figcaption>
+                           <b>Figure 2:</b> Plotting the OD<sub>600</sub> of <i>E. coli</i> DH5alpha each carrying different plasmid  versus cultivation time in a volume of 1 mL LB medium containing 0 mM CuSO<sub>4</sub>. Experiments were performed in a 24-well plate at 37 °C and 350 rpm (n=3).
+                      </figcaption>
+                   </figure>
-<button onclick="myFunction()" class="refbtn"> References &#9662;</button>
+<article>
+The growth of strains carrying different constructs in absence of cupric salt was examined. No significant difference between the highest OD<sub>600</sub> values of the cells could be observed. However, cells carrying plasmids under the control of the pBAD promoter showed a different growth pattern since they can rely on another carbon source once glucose is metabolized. This leads to two consecutive exponential phases resulting in similar OD<sub>600</sub> values compared to those only relying on glucose as a carbon source. Other than that, no significant changes in growth behavior were observed.
+</article>
-                  <div id="myDIV" class="reftext">
+<figure role="group">
+                      <img class="figure hundred" src="https://static.igem.org/mediawiki/parts/0/01/T--Bielefeld-CeBiTec--jr--4_mM_CuSO4.png">
+                      <figcaption>
+                           <b>Figure 3:</b> Plotting the OD<sub>600</sub> of <i>E. coli</i> DH5alpha each carrying different plasmids versus time in a volume of 1 mL LB medium containing 4 mM CuSO<sub>4</sub>. Experiments were performed in a 24-well plate at 37 °C and 350 rpm (n=3).
+                      </figcaption>
+                   </figure>
-<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>
+<article>
+No mutant carrying any of the BioBricks exhibits an advantage regarding the growth rate or the final OD<sub>600</sub> value. While BBa_K2638110 and BBa_K2638114 exhibit the same growth pattern as the mutant carrying an empty pSB1C3 vector, BBa_K2638112 and BBa_K2638109 showed significant lower growth rates and did not reach an OD<sub>600</sub> value as high as the other mutants. The construct BBa_K2638118 achieved an OD<sub>600</sub> value of 0.79.
+</article>
-<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>
+<figure role="group">
+                      <img class="figure hundred" src="https://static.igem.org/mediawiki/parts/6/6b/T--Bielefeld-CeBiTec--jr--8_mM_CuSO4.png">
+                      <figcaption>
+                           <b>Figure 4:</b> Plotting the OD of <i>E. coli</i> DH5alpha each carrying different plasmid versus time in a volume of 1 mL LB medium containing 8 mM CuSO<sub>4</sub>. Experiments were performed in a 24-well plate at 37 °C and 350 rpm (n=3).
+                      </figcaption>
+                   </figure>
-<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>
+<article>
+Every mutant showed visible growth only during the first two hours at best. Afterwards, there was no significant increase in the OD<sub>600</sub> value. The highest OD<sub>600</sub> values were achieved by the construct BBa_K2638112, the lowest OD<sub>600</sub> values by the construct BBa_K2638109.</br>
+However, since no growth improving effect using our initial approaches against toxicity  could be observed, we decided to conduct an experiment based on the viability of the cells at the measured points of time at the beginning of the experiment and after three, six and 9 hours of cultivation.</br>
+We grew <i>E. coli</i> containing the constructs BBa_K2638112, BBa_K2638110, BBa_K2638110, BBa_K2638118 and pSB1C3 in 10 ml LB medium supplemented with 30 ng/ml chloramphenicol for 9 hours and either 0 mM, 4 mM or 8 mM CuSO<sub>4</sub>. Every three hours, a sample was taken and 50 µl were plated in the following dilutions: 10<sup>-4</sup>, 10<sup>-5</sup> and 10<sup>-6</sup>. Additionally, whenever a sample was taken, the optical density at 600 nm was measured.</br>
+In order to validate our results, we correlated the optical density and the determined CFU (Colony Forming Units).
+</article>
-<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>
+<figure role="group">
+                      <img class="figure hundred" src="https://static.igem.org/mediawiki/parts/1/1c/T--Bielefeld-CeBiTec--jr--0_mM_CFU.png">
+                      <figcaption>
+                           <b>Figure 5:</b> Plotting of the optical density at 600 nm of cells grown in LB medium containing 30 ng/ml chloramphenicol and no cupric salt at 37 °C against the amount of colony forming units (CFU) in ml<sup>-1</sup>, as well as linear fits (n=3).
+                      </figcaption>
+                   </figure>
+<article>
+The OD<sub>600</sub> clearly correlates with the amount of colony forming units (CFU) when no further cupric sulfate was added to the medium. The Pearson correlation coefficient R of the different constructs can be found in the table below, indicating a significant positive correlation between the two values. The adjoined coefficients of determination R<sup>2</sup> implicate that at least 90 % of all changes in the optical density result in a change in the amount of colony forming units. This value increases even further regarding the different implemented constructs to at least 97.3 %.
+</article>
-<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>
+<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>
+			<th>0.996</th>
+			<th>0.952</th>
+			<th>0.993</th>
+			<th>0.987</th>
+			<th>0.994</th>
+		  </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>
+<article>
+The same measurements were performed for cells at cupric sulfate concentrations of 4 mM and 8 mM. The linear fits for the constructs grown in both supplemented media are shown in figures 6 and 7.
+</article>
-<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>
+<figure role="group">
+                      <img class="figure hundred" src="https://static.igem.org/mediawiki/parts/f/f9/T--Bielefeld-CeBiTec--jr--4_mM_CFU.png">
+                      <figcaption>
+                           <b>Figure 6:</b> Plotting of the optical density at 600 nm of cells grown in LB medium containing 30 ng/ml chloramphenicol and 4 mM CuSO<sub>4</sub> at 37 °C against the amount of colony forming units (CFU) in ml<sup>-1</sup>, as well as linear fits (n=3).
+                      </figcaption>
+                   </figure>
-<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>
+<article>
+A positive correlation was indicated by determining the Pearson correlation coefficient and the coefficient for determination for the different constructs. However, according to its coefficient of determination, in the case of BBa_K2638110 only 83.7 % of all changes of the optical density could be referred to the changes in the amount of colony forming units,. according to its coefficient of determination. In general, a positive correlation between the optical density and the CFU could be clearly determined.
+</article>
+<table id="t01" class="centern" style="margin-top:30px; margin-bottom:30px;">
+ <caption style="line-height:1.5; text.align:left;"><b>Table 2:</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>
+			<th>0.953</th>
+			<th>0.998</th>
+			<th>0.968</th>
+			<th>0.972</th>
+			<th>0.915</th>
+		  </tr>
+		  <tr>
+			<th>R<sup>2</sup> value<th>
+			<td>0.908</td>
+			<td>0.997</td>
+			<td>0.937</td>
+			<td>0.945</td>
+			<td>0.837</td>
+		  </tr>
+		</table>
+<article>
+In contrast to those cells grown in the absence of cupric sulfate and in the presence of 4 mM cupric sulfate, no positive correlation between the optical density and the CFU could be observed. Instead, the cells grown in LB medium with 8 mM cupric sulfate were proven to show a negative correlation. This is clearly visible in figure 7 (see below).
+</article>
-<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>
+<figure role="group">
+                      <img class="figure hundred" src="https://static.igem.org/mediawiki/parts/e/e5/T--Bielefeld-CeBiTec--jr--8_mM_CFU.png">
+                      <figcaption>
+                           <b>Figure 7:</b> Plotting of the optical density at 600 nm of cells grown in LB medium containing 30 ng/ml chloramphenicol and 8 mM CuSO<sub>4</sub> at 37 °C against the amount of colony forming units (CFU) in ml<sup>-1</sup>, as well as linear fits (n=3).
+                      </figcaption>
+                   </figure>
-<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>
+<article>
+The negative correlation was revealed by Pearson correlation coefficient R and coefficient of determination R<sup>2</sup>.
-<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>
+</article>
+<table id="t01" class="centern" style="margin-top:30px; margin-bottom:30px;">
+  <caption style="line-height:1.5; text.align:left;"><b>Table 2:</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>
+			<th>-0.576</th>
+			<th>-0.955</th>
+			<th>-0.817</th>
+			<th>-0.952</th>
+			<th>-0.924</th>
+		  </tr>
+		  <tr>
+			<th>R<sup>2</sup> value<th>
+			<td>0.332</td>
+			<td>0.913</td>
+			<td>0.668</td>
+			<td>0.906</td>
+			<td>0.853</td>
+		  </tr>
+		</table>
+<article>
+R2 was less than half as big as the R2 values of the other constructs and of all constructs at different concentrations of cupric sulfate. The negative correlation was likely due to cells dying at higher copper concentrations but not being degraded immediately so that the OD600 value suggests a higher cell density than viability. Therefore, the correlation of every construct shifts to the negative area. Furthermore, the highest R and R2 values have been determined for the mutant carrying the empty pSB1C3 vector.
+</article>
+<figure role="group">
+                      <img class="figure hundred" src="https://static.igem.org/mediawiki/parts/5/51/T--Bielefeld-CeBiTec--jr--CFU_0_and_4.jpeg">
+                      <figcaption>
+                           <b>Figure 8:</b> Bar chart of the CFUs of the tested constructs in LB medium supplemented with either 0 mM or 4 mM cupric sulfate at 37 °C (n=3).
+                      </figcaption>
+                   </figure>
+<article>
+No significant difference in growth or viability in comparison to the empty pSB1C3 vector was observed for any tested construct at a concentration of either 0 mM or 4 mM cupric sulfate. According to the previously recorded growth trends, the growth rate decreased visibly with a rising concentration of copper ions. However, since no effect significantly improving the amount of colony forming units compared to the wildtype could be determined for these concentrations, the CFU at 8 mM cupric sulfate was evaluated.
+</article>
+<figure role="group">
+                      <img class="figure hundred" src="https://static.igem.org/mediawiki/parts/6/6c/T--Bielefeld-CeBiTec--jr--CFU8mM.jpeg">
+                      <figcaption>
+                           <b>Figure 9:</b> Bar chart of the CFUs of the tested constructs in LB medium supplemented with 8 mM cupric sulfate at 37 °C (n=3), as well as the assigned significance levels to explain which constructs differ significantly from each other.
+                      </figcaption>
+                   </figure>
+<article>
+The number of colony forming units at the beginning of the measurement, after 0 hours, already shows indicates a great difference. Furthermore, the CFU in the course of time reveals differences for certain constructs in comparison to the wildtype. BBa_K2638112 and BBa_K2638118 appear to be more viable after 3, 6 and 9 hours of cultivation in 8 mM cupric sulfate containing medium. However, since no growth appeared to occur, the evaluation of this data concentrates on the preservation of the existing biomass rather than to improve its growth capabilities. After all, the cells are meant to be incubated and not cultivated in our remediation process of filtering water.
+ </br>To examine the collected data regarding the influence of time and tested constructs, we conducted a two-factor ANOVA (analysis of variance) with repeated measurements for both factors.
+The aim is to discard the null hypothesis stating that there is no significant difference between the temporal developments of the tested constructs. Therefore, we conducted Mauchly’s Test of Sphericity to determine the samples sphericity. The test suggested highly with high significance to accept the assumption of sphericity with a significance level of .000 meaning that a correction of values by Greenhouse-Geisser was needed. The test of Within-Subject Effects resulted in the conclusion that the applied construct as well as the combination of the temporal development and the construct has a significant influence on the amount of colony forming units. In case of the construct, the influence was determined to be even highly significant. The constructs, the temporal development and the combination of those factors were examined, and each assigned to a significance level.</br>
+In conclusion, we were able to develop two composite parts which increase a cell’s tolerance for elevated concentrations of copper. This ameliorated tolerance could be verified for concentrations up to 8 mM of CuSO4 for a duration of up to 9 hours. This is a great first approach to realize our project and enable our cells to survive longer in the mining drainage. As our composite can also be used by iGEM teams in the future, we deem these results to be very useful to the community. Further applications could also be in the remediation of contaminated soils and seas, as well as industrial applications and processes in a heavy metal rich environment.
+</article>
                     </div>