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

Revision as of 18:03, 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.
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 <h2>Toxicity assay</h2>
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+<div class="article">
 As intracellular copper triggers toxic effects on the cell (also see <a href="https://2018.igem.org/Team:Bielefeld-CeBiTec/Toxicity_Theory">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:
-</article>
+</div>
 <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>Parts used in toxicity assay (growth curves)</caption>