Difference between revisions of "Part:BBa K2638001"
 
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CopC is a gene that encodes for a soluble protein within the periplasmic space (β-barrel structure) with a highly specific Cu(I) and another highly specific Cu(II) binding site. It is part of the CopABCD gene cluster. CopC in Pseudomonas syringae pathovar tomato promotes Cu(II) uptake into the periplasmic space. It was used a homologue from Pseudomonas brassicacearum 3Re2-7 13. Together with CopD (BBa_K2638002) it promotes copper uptake from outside the cell into the cytoplasm.  
 
CopC is a gene that encodes for a soluble protein within the periplasmic space (β-barrel structure) with a highly specific Cu(I) and another highly specific Cu(II) binding site. It is part of the CopABCD gene cluster. CopC in Pseudomonas syringae pathovar tomato promotes Cu(II) uptake into the periplasmic space. It was used a homologue from Pseudomonas brassicacearum 3Re2-7 13. Together with CopD (BBa_K2638002) it promotes copper uptake from outside the cell into the cytoplasm.  
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<h2>Short Summary</h2>
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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 intended, we conducted several experiments analyzing Cu(II) ion uptake. We conducted growth experiments and observed hindered cell growth when expressing our transporter proteins, which indicates import of toxic copper into the cell. Furthermore, we measured membrane permeability thereby, showing the ion channel nature of the transporter proteins in the outer membrane. Finally we showed the proteins specifity for taking up Cu(II).</article>
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<h2>Toxicity Assays</h2>
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As intracellular copper leads to toxic effects in the cell (read more on our <a href="http://2018.igem.org/Team:Bielefeld-CeBiTec/Toxicity_Theory" target="_blank">toxicity</a>) page, an increased uptake of Cu(II) ions should slow down and exacerbate cell growth. Therefore, we examined the growth of <i>E. coli</i> KRX expressing <i>copC</i>, <i>copD</i>, <i>oprC</i> and <i>hmtA</i> in lysogeny broth (LB) media with 30 ng/µL chloramphenicol supplemented with different concentrations of CuSO<sub>4</sub> (0 mM, 1 mM, 2 mM, 3 mM, 4 mM, 8 mM) by measuring the optical density at a wavelength of 600 nm (OD600). As a control we decided to use the pSB1C3 null vector under same conditions as well. 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;). For expression of the BioBricks either a T7 promoter and a ribosome binding site (RBS) (BBa_K525998) or a combination of the <i>pBAD/araC</i> promoter (BBa_I0500) and a RBS (BBa_B0030) (RBS) were cloned upstream of <i>copC</i> (BBa_K2638001), <i>copD</i> (BBa_K2638002), (<i>oprC</i> (BBa_K2638200) and <i>hmtA</i> (BBa_K2638000). The resulting parts are shown in table 1:
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Latest revision as of 03:58, 18 October 2018


CopC coding sequence

CopC is a gene that encodes for a soluble protein within the periplasmic space (β-barrel structure) with a highly specific Cu(I) and another highly specific Cu(II) binding site. It is part of the CopABCD gene cluster. CopC in Pseudomonas syringae pathovar tomato promotes Cu(II) uptake into the periplasmic space. It was used a homologue from Pseudomonas brassicacearum 3Re2-7 13. Together with CopD (BBa_K2638002) it promotes copper uptake from outside the cell into the cytoplasm.

Short Summary

To test whether our accumulation system with the importers oprC, hmtA, copC and copD works as intended, we conducted several experiments analyzing Cu(II) ion uptake. We conducted growth experiments and observed hindered cell growth when expressing our transporter proteins, which indicates import of toxic copper into the cell. Furthermore, we measured membrane permeability thereby, showing the ion channel nature of the transporter proteins in the outer membrane. Finally we showed the proteins specifity for taking up Cu(II).

Toxicity Assays

As intracellular copper leads to toxic effects in the cell (read more on our toxicity) page, an increased uptake of Cu(II) ions should slow down and exacerbate cell growth. Therefore, we examined the growth of E. coli KRX expressing copC, copD, oprC and hmtA in lysogeny broth (LB) media with 30 ng/µL chloramphenicol supplemented with different concentrations of CuSO4 (0 mM, 1 mM, 2 mM, 3 mM, 4 mM, 8 mM) by measuring the optical density at a wavelength of 600 nm (OD600). As a control we decided to use the pSB1C3 null vector under same conditions as well. The measurement was performed with the Infinite® 200 PRO in a 24 wellplate with flat bottom (Greiner®). For expression of the BioBricks either a T7 promoter and a ribosome binding site (RBS) (BBa_K525998) or a combination of the pBAD/araC promoter (BBa_I0500) and a RBS (BBa_B0030) (RBS) were cloned upstream of copC (BBa_K2638001), copD (BBa_K2638002), (oprC (BBa_K2638200) and hmtA (BBa_K2638000). The resulting parts are shown in table 1:

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 304
    Illegal AgeI site found at 358
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 246