Difference between revisions of "Team:Bielefeld-CeBiTec/Basic Part"

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                 <div class="title">Basic Parts</div>
 
                 <div class="title">Basic Parts</div>
 +
     
 +
      <h2>Best New Basic Part</h2>
 +
      <div class="article">Our best new basic part is <a href="http://parts.igem.org/Part:BBa_K2638002" target="_blank">BBa_K2638002</a>, <i>copD</i>, an inner membrane importer for copper ions. It is part of our <a href="https://2018.igem.org/Team:Bielefeld-CeBiTec/Accumulation target="_blank">accumulation system</a> in <i>E. coli</i>. In its native organism <i>Pseudomonas brassicacearum</i> 3RE2-7 it is expressed next to <i>copC</i>, which supports effective uptake.
 +
</div>
  
−
  <article>For our metal resource recovery system we build lots of new biobricks for accumulation and storage of metal ions, toxicity counteractions and nanoparticle formation. We also designed Parts for our siRNA expression and analyzation tool to regulate expression of individual genes on RNA level.
+
<div="article">We expressed the gene in two different expression systems, one with a T7 promoter and RBS (<a href="http://parts.igem.org/Part:BBa_K2638004" target="_blank">BBa_K2638004</a>) and the other with a <i>pBAD/araC</i> and RBS system (<a href="http://parts.igem.org/Part:BBa_K2638006" target="_blank">BBa_K2638006</a>). All results also in comparison with the other importers we characterized can be found on our <a href="https://2018.igem.org/Team:Bielefeld-CeBiTec/Accumulation_Results" target="_blank">accumulation results</a> page.</div>
  
 +
<article>We conducted growth experiments showing inhibited growth in the presence of Cu(II) ions from increased toxicity of imported copper (figure 1 and figure 2).With expression of <i>copD</i> (BBa_K2638004) decreased growth upon expression but also slightly higher sensitivity towards copper could be observed. Furthermore, the effect of cell death was detected again, although the reaction is hardly measurable with a fast recovery of the cells after about 100 minutes. With <i>copD</i> in combination with pBAD/araC/RBS (BBa_K2638006) this can be reproduced and the effect there is more obvious (see figure 2).</article>
 +
 +
<figure role="group">
 +
                      <img class="figure hundred" src="https://static.igem.org/mediawiki/2018/4/4b/T--Bielefeld-CeBiTec--ES--Growth_Curve_BBa_K2638004.png">
 +
 +
<figcaption>
 +
                          <b>Figure 1: </b>Growth curves of <i>E. coli</i> KRX BBa_K2638004 at different CuSO<sub>4</sub> concentrations. Left: No induction. Right: Induction started simultaneously with inoculation with 0.1 % rhamnose and 0.1 mM IPTG.
 +
                      </figcaption>
 +
                  </figure>
 +
                 
 +
                 
 +
<figure role="group">
 +
                      <img class="figure hundred" src="https://static.igem.org/mediawiki/2018/e/e5/T--Bielefeld-CeBiTec--ES--Growth_Curve_BBa_K2638006.png">
 +
 +
<figcaption>
 +
                          <b>Figure 2: </b>Growth curves of <i>E. coli</i> DH5&alpha; BBa_K2638006 at different CuSO<sub>4</sub> concentrations. Left: No induction. Right: Induction started simultaneously with inoculation with 1 % arabinose.
 +
                      </figcaption>
 +
                  </figure>
 +
                 
 +
 +
<article>Using the growth curve data from <i>E. coli</i>DH5&alpha; carrying BBa_K2638006 we also compared relative growth of a strain at 2 mM or 3 mM Cu(II) against the growth at 0 mM Cu(II) for not induced and induced cultures (figure 3). The data shows that supplementary copper even benefits growth at low concentrations. At small copper concentrations non-induced cells grow even faster then without added CuSO<sub>4</sub> (9.9 % with 2 mM Cu(II) and 21.2 % with 3 mM Cu(II)). Upon induction with arabinose this changes and growth is inhibited by 21.5% &plusmn; 3.3% at 2 mM Cu(II) and 42 % &plusmn; 5.4 % with 3 mM Cu(II). This is a strong indicator for successful copper uptake.</article>
 +
 +
<figure role="group">
 +
                      <img class="figure eighty" src="https://static.igem.org/mediawiki/2018/9/9f/T--Bielefeld-CeBiTec--ES--Growth_Comparison_CopD.png">
 +
 +
<figcaption>
 +
                          <b>Figure 3: </b>Relative OD 600 of not induced cultures growing with 0 mM, 2 mM or 3 mM supplementary Cu(II) in comparison with 0 mM Cu(II) (n.i. 0 mM, n.i. 2 mM, n.i. 3 mM). The other curves (i. 2 mM, i. 3 mM) of with arabinose induced cultures are compared to cultures grown with 0 mM arabinose and 1 % arabinose.
 +
                      </figcaption>
 +
                  </figure>
 +
 +
<h2>Membrane Permeability Assays</h2>
 +
 +
<article>
 +
1-N-phenylnaphthylamine membrane-permeabilization (NPN) assays are a fast method to measure the permeability of outer cell membranes.
 +
The NPN assays were all performed under the same conditions. The cells were either induced with  0.1 % rhamnose and 0.1 mM IPTG or with 1 % arabinose. Afterwards, fluorescence was measured in the range from 380 – 550 nm after exitation with light with the wavelength of 355 nm. The fluorescence values for every measurement were normalized to the fluorescence value at 382 nm.</article>
 +
 +
<div class="article">
 +
In both strains which expressed the <i>copD</i> composite parts BBa_2638004 (T7 promoter, RBS and <i>copD</i>) and BBa_K2638006 (<i>pBAD/araC</i> promoter, RBS and <i>copD</i>) a higher increase in the percental fluorescence increase was shown compared to the pSB1C3 control strain (yellow curve figure 3).
 +
The strain expressing <i>copD</i> under control of the <i>pBAD/araC</i> promoter with RBS (<a href="http://parts.igem.org/Part:BBa_K2638006" target="_blank">BBa_K2638006</a>, blue curve in figure 3) showed a maximum of 94.16 &plusmn; 10.89 % in the fluorescence emission at 408 nm. This is an increase of 59.03 % to the empty control vector pSB1C3.
 +
The <i>copD</i> strain with the T7 promoter (<a href="http://parts.igem.org/Part:BBa_K2638004" target="_blank">BBa_2638004</a>, red curve in figure 3) showed a maximum regarding the fluorescence of 68.10 &plusmn; 2.84 % at 408 nm. This is an increase of 32.99 % in comparison to the  empty vector control.
 +
The difference increased fluorescence at 408 nm of both <i>copD</i> expressing strains compared to the empty vector control show a substantial increased of the membrane permeability of <i>E. coli</i>. This also indicates substantial uptake.
 +
</div>
 +
 +
<figure role="group">
 +
                      <img class="figure hundred" src="https://static.igem.org/mediawiki/2018/1/15/T--Bielefeld-CeBiTec--JZ--FluorescenceNPNCopD.jpeg">
 +
 +
<figcaption>
 +
                          <b>Figure 3: </b>Relative fluorescence increase of <i>E. coli</i> KRX strains with BBa_K2638004 (<i>copD</i> containing plasmid with T7 promoter control, red curve), BBa_K2638006 (<i>copD</i> containing plasmid with <i>pBAD/araC</i> promoter with RBS control, blue curve) and as an empty vector control an empty pSB1C3 in <i>E. coli</i> KRX (yellow curve). The excitation wavelenght is 355 nm. The OD<sub>600</sub> of cells was adjusted with the NPN stock solution to the same value. 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&reg;).
 +
                      </figcaption>
 +
                  </figure>
 +
                 
 +
<article>Specifity of Cu(II) uptake is shown with an ONPG-assay (<i>orhto</i>-Nitrophenyl-&beta;-galactoside assay). The ONPG assay shows no unspecific uptake for <i>copD</i> and the other analyzed constructs (figure 4).</article>
 +
 +
<figure role="group">
 +
                      <img class="figure hundred" src="https://static.igem.org/mediawiki/2018/2/29/T--Bielefeld-CeBiTec--ES--ONPG.png">
 +
 +
<figcaption>
 +
                          <b>Figure 4: </b>Results of the ONPG assay. The absorption spectra are measured at &lambda; = 400 nm over <i>t</i> = 3000 s. 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&reg;).
 +
                      </figcaption>
 +
                  </figure>
 +
 +
<article>For a complete discussion look up the <a href="https://2018.igem.org/Team:Bielefeld-CeBiTec/Accumulation_Results" target="_blank">results page</a></article>
 +
 +
<article>In general our data shows that our copper uptake Biobricks work as expected. The import of copper can be shown by increasing toxic effects on cells, as the growth curves indicate an obvious growth inhibition for OprC and CopD.  The CopD encoding Biobricks BBa_K2638002, BBa_K2638004 and BBa_K2638006 show the most impressive results both in the toxicity test and the NPN assay. This result was expected, as the import of Cu(II) occurs actively across the inner cell membrane into the cytoplasm (Lawton <i>et al.</i>, 2016), where toxic effects have the strongest impact.</article>
 +
 +
<article>Further interesting experiments would be the combination of OprC (BBa_K2638200), CopC (BBa_K2638001) with CopD (BBa_2638002) in one biobrick. The combination of an importer into the periplasm and into the cytoplasm could improve the effectivity of uptake dramatically. To further support CopD, CopC could deliver Cu(II) to the transporter through the periplasm. The already performed growth experiments could be performed and measurements of intracellular copper may be conducted.
 +
</article>
 +
 +
  <h2>Collection of Basic Parts</h2>
 +
 
 +
  <article>For our metal resource recovery system we build lots of new biobricks for accumulation and storage of metal ions, toxicity counteractions and nanoparticle formation. We also designed parts for our siRNA expression and analyzation tool to regulate expression of individual genes on RNA level.
 
                   </article>
 
                   </article>
  
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             <td>Antonin Lenzen</td>
 
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            <td>Antonin Lenzen</td>
 
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<button onclick="myFunction()" class="refbtn"> References &#9662;</button>
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                  <div id="myDIV" class="reftext">
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<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).</br>
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Revision as of 02:58, 18 October 2018

Basic Parts

Best New Basic Part

Our best new basic part is BBa_K2638002, copD, an inner membrane importer for copper ions. It is part of our accumulation system in E. coli. In its native organism Pseudomonas brassicacearum 3RE2-7 it is expressed next to copC, which supports effective uptake.
We expressed the gene in two different expression systems, one with a T7 promoter and RBS (BBa_K2638004) and the other with a pBAD/araC and RBS system (BBa_K2638006). All results also in comparison with the other importers we characterized can be found on our accumulation results page.
We conducted growth experiments showing inhibited growth in the presence of Cu(II) ions from increased toxicity of imported copper (figure 1 and figure 2).With expression of copD (BBa_K2638004) decreased growth upon expression but also slightly higher sensitivity towards copper could be observed. Furthermore, the effect of cell death was detected again, although the reaction is hardly measurable with a fast recovery of the cells after about 100 minutes. With copD in combination with pBAD/araC/RBS (BBa_K2638006) this can be reproduced and the effect there is more obvious (see figure 2).
Figure 1: Growth curves of E. coli KRX BBa_K2638004 at different CuSO4 concentrations. Left: No induction. Right: Induction started simultaneously with inoculation with 0.1 % rhamnose and 0.1 mM IPTG.
Figure 2: Growth curves of E. coli DH5α BBa_K2638006 at different CuSO4 concentrations. Left: No induction. Right: Induction started simultaneously with inoculation with 1 % arabinose.
Using the growth curve data from E. coliDH5α carrying BBa_K2638006 we also compared relative growth of a strain at 2 mM or 3 mM Cu(II) against the growth at 0 mM Cu(II) for not induced and induced cultures (figure 3). The data shows that supplementary copper even benefits growth at low concentrations. At small copper concentrations non-induced cells grow even faster then without added CuSO4 (9.9 % with 2 mM Cu(II) and 21.2 % with 3 mM Cu(II)). Upon induction with arabinose this changes and growth is inhibited by 21.5% ± 3.3% at 2 mM Cu(II) and 42 % ± 5.4 % with 3 mM Cu(II). This is a strong indicator for successful copper uptake.
Figure 3: Relative OD 600 of not induced cultures growing with 0 mM, 2 mM or 3 mM supplementary Cu(II) in comparison with 0 mM Cu(II) (n.i. 0 mM, n.i. 2 mM, n.i. 3 mM). The other curves (i. 2 mM, i. 3 mM) of with arabinose induced cultures are compared to cultures grown with 0 mM arabinose and 1 % arabinose.

Membrane Permeability Assays

1-N-phenylnaphthylamine membrane-permeabilization (NPN) assays are a fast method to measure the permeability of outer cell membranes. The NPN assays were all performed under the same conditions. The cells were either induced with 0.1 % rhamnose and 0.1 mM IPTG or with 1 % arabinose. Afterwards, fluorescence was measured in the range from 380 – 550 nm after exitation with light with the wavelength of 355 nm. The fluorescence values for every measurement were normalized to the fluorescence value at 382 nm.
In both strains which expressed the copD composite parts BBa_2638004 (T7 promoter, RBS and copD) and BBa_K2638006 (pBAD/araC promoter, RBS and copD) a higher increase in the percental fluorescence increase was shown compared to the pSB1C3 control strain (yellow curve figure 3). The strain expressing copD under control of the pBAD/araC promoter with RBS (BBa_K2638006, blue curve in figure 3) showed a maximum of 94.16 ± 10.89 % in the fluorescence emission at 408 nm. This is an increase of 59.03 % to the empty control vector pSB1C3. The copD strain with the T7 promoter (BBa_2638004, red curve in figure 3) showed a maximum regarding the fluorescence of 68.10 ± 2.84 % at 408 nm. This is an increase of 32.99 % in comparison to the empty vector control. The difference increased fluorescence at 408 nm of both copD expressing strains compared to the empty vector control show a substantial increased of the membrane permeability of E. coli. This also indicates substantial uptake.
Figure 3: Relative fluorescence increase of E. coli KRX strains with BBa_K2638004 (copD containing plasmid with T7 promoter control, red curve), BBa_K2638006 (copD containing plasmid with pBAD/araC promoter with RBS control, blue curve) and as an empty vector control an empty pSB1C3 in E. coli KRX (yellow curve). The excitation wavelenght is 355 nm. The OD600 of cells was adjusted with the NPN stock solution to the same value. The measurement was performed with the Infinite® 200 PRO in a 24 wellplate with flat bottom (Greiner®).
Specifity of Cu(II) uptake is shown with an ONPG-assay (orhto-Nitrophenyl-β-galactoside assay). The ONPG assay shows no unspecific uptake for copD and the other analyzed constructs (figure 4).
Figure 4: Results of the ONPG assay. The absorption spectra are measured at λ = 400 nm over t = 3000 s. The measurement was performed with the Infinite® 200 PRO in a 24 wellplate with flat bottom (Greiner®).
In general our data shows that our copper uptake Biobricks work as expected. The import of copper can be shown by increasing toxic effects on cells, as the growth curves indicate an obvious growth inhibition for OprC and CopD. The CopD encoding Biobricks BBa_K2638002, BBa_K2638004 and BBa_K2638006 show the most impressive results both in the toxicity test and the NPN assay. This result was expected, as the import of Cu(II) occurs actively across the inner cell membrane into the cytoplasm (Lawton et al., 2016), where toxic effects have the strongest impact.
Further interesting experiments would be the combination of OprC (BBa_K2638200), CopC (BBa_K2638001) with CopD (BBa_2638002) in one biobrick. The combination of an importer into the periplasm and into the cytoplasm could improve the effectivity of uptake dramatically. To further support CopD, CopC could deliver Cu(II) to the transporter through the periplasm. The already performed growth experiments could be performed and measurements of intracellular copper may be conducted.

Collection of Basic Parts

For our metal resource recovery system we build lots of new biobricks for accumulation and storage of metal ions, toxicity counteractions and nanoparticle formation. We also designed parts for our siRNA expression and analyzation tool to regulate expression of individual genes on RNA level.
Identifier Type Description Designer Length
BBa_K2638001 Coding CopC coding sequence Erika Schneider 387
BBa_K2638002 Coding CopD CDS Erika Schneider 912
BBa_K2638000 Coding Heavy Metal Transporter (hmtA) Erika Schneider 1920
BBa_K2638100 Coding alkyl hydroperoxide reductase, C22 subunit (ahpC) Johannes Ruhnau 564
BBa_K2638101 Coding alkyl hydroperoxide reductase, F52a subunit (ahpF) Johannes Ruhnau 1566
BBa_K2638103 Coding glutathione synthetase (gshB) Johannes Ruhnau 951
BBa_K2638105 Coding catalase HPI (katG) Johannes Ruhnau 2181
BBa_K2638106 Coding superoxide dismutase (sodA) Johannes Ruhnau 621
BBa_K2638120 Coding glutathione peroxidase (btuE) Johannes Ruhnau 552
BBa_K2638121 Coding glutathione reductase (gor) Johannes Ruhnau 1353
BBa_K2638150 Coding Glutathione gamma-glutamylcysteinyltransferase 1 Matthias Otto 1458
BBa_K2638151 Coding Glutathione gamma-glutamylcysteinyltransferase 1 with RBS and pTet promoter Matthias Otto 1535
BBa_K2638152 Coding Glutathione gamma-glutamylcysteinyltransferase 1 with RBS and Anderson promoter Matthias Otto 1519
BBa_K2638153 Coding Glutathione gamma-glutamylcysteinyltransferase 1 with AraBAD promoter and RBS Matthias Otto 2694
BBa_K2638200 Coding OprC (TonB dependent copper transport porin) Jakob Zubek 2127
BBa_K2638426 Reporter mRFP+ Levin Joe Klages 827
BBa_K2638601 Coding Human Ferritin Heavy Chain Copper Enhanced Binding Christoph Geske 552
BBa_K2638602 Coding Human Ferritin Heavy Chain E-Helix Removed Weaker Binding Christoph Geske 480
BBa_K2638603 Coding Human Ferritin Heavy Chain Copper Enhanced Binding and E-Helix removed Christoph Geske 474
BBa_K2638701 Generator Seamless siRNA Expression Vektor pGuide Antonin Lenzen 1780
BBa_K2638702 Generator Seamless siRNA Expression Vektor for Hfq binding siRNAs Antonin Lenzen 1865
BBa_K2638707 Reporter siRNA Target Vector with BFP and a GGGGS Linker Antonin Lenzen 2779
BBa_K2638708 Reporter siRNA Target Vector with BFP and an EAAAK Linker Antonin Lenzen 2779
BBa_K2638709 Reporter siRNA Target Vector with BFP and a cMyc Linker Antonin Lenzen 2779
BBa_K2638710 Reporter siRNA Target Vector with BFP and XP Linker Antonin Lenzen 2779
BBa_K2638711 Reporter siRNA Target Vector with AmilCP and a GGGGS Linker Antonin Lenzen 1226
BBa_K2638712 Reporter siRNA Target Vector with AmilCP and EAAAK Linker Antonin Lenzen 1226
BBa_K2638713 Reporter siRNA Target Vector with AmilCP and a cMyc Linker Antonin Lenzen 1226
BBa_K2638714 Reporter siRNA Target Vector with AmilCP and XP Linker Antonin Lenzen 1226
BBa_K2638752 Regulatory pLac including a TetO sequence in place of the CAP binding sequence Antonin Lenzen 206
BBa_K2638753 Regulatory pLac including a Tet operator sequence behind the Lac operator Antonin Lenzen 213
BBa_K2638754 Coding E. coli Bacterioferritin Bfr Antonin Lenzen 477
BBa_K2638755 Coding Ferredoxin reductase Antonin Lenzen 747
BBa_K2638756 Coding Bacterial non-heme ferritin FtnA from Escherichia coli Antonin Lenzen 498
BBa_K2638757 Coding Ferric Uptake Regulator (Fur) from Escherichia coli K12 Antonin Lenzen 447
BBa_K2638758 Generator Seamless siRNA Expression Vektor for Omp-Utr protected siRNAs Antonin Lenzen 1897
BBa_K2638759 Generator Seamless siRNA Expression Vektor with an Hfq binding tag and OmpA 5'-UTR Antonin Lenzen 1982
BBa_K2638990 Coding Mutated Human Ferritin Heavy Chain (Backup) Vanessa Krämer 549
BBa_K2638999 Coding Mutated Human Ferritin Heavy Chain Vanessa Krämer 552


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).