Difference between revisions of "Part:BBa K2638200"
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==Results== | ==Results== | ||
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<h2>Toxicity Assays</h2> | <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>oprC</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®). 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>oprC</i> (BBa_K2638200). | + | 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>oprC</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"> |
| + | <body>Infinite® 200 PRO</a> 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 <i>pBAD/araC</i> promoter (BBa_I0500) and a RBS (BBa_B0030) (RBS) were cloned upstream of (<i>oprC</i> (BBa_K2638200). | ||
<article>All toxicity measurements were performed by inoculating <i>E. coli</i> KRX cultures harbouring the plasmid of interest with a starting OD600 of 0.01 from an overnight culture. Afterwards cells were grown (37 °C, 350 rpm) for 3 h and were induced either with 0.1 % rhamnose and 0.1 mM IPTG for the T7 constructs or 1 % arabinose for the arabinose constructs. The OD600 of the cultures were measured every hour for up to 12 hours. | <article>All toxicity measurements were performed by inoculating <i>E. coli</i> KRX cultures harbouring the plasmid of interest with a starting OD600 of 0.01 from an overnight culture. Afterwards cells were grown (37 °C, 350 rpm) for 3 h and were induced either with 0.1 % rhamnose and 0.1 mM IPTG for the T7 constructs or 1 % arabinose for the arabinose constructs. The OD600 of the cultures were measured every hour for up to 12 hours. | ||
Revision as of 03:54, 18 October 2018
OprC (TonB dependent copper transport porin)
OprC is an outer membrane porin which binds and transports specifically copper. It belongs to the the superfamily of the TonB-dependent transporters. This variant of OprC was taken from Pseudomonas brassicacearum 3Re2-7 13, a not yet published strain.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 252
Illegal NgoMIV site found at 583
Illegal NgoMIV site found at 787
Illegal NgoMIV site found at 1831
Illegal NgoMIV site found at 2032
Illegal AgeI site found at 246 - 1000COMPATIBLE WITH RFC[1000]