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

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<article>The growth of the T7 RBS strains expressing either <i>copC</i>, <i>copD</i>, <i>oprC</i> or <i>hmtA</i>after induction with 0.1 % rhamnose and 0.1 mM IPTG in comparison with non-induced cells is reduced at all tested Cu(II) concentrations in the medium.
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Figure 1 shows the growth of <i>E. coli</i> DH5a with BBa_K2638201 (<i>oprC</i>). The right graph shows the growth after induction in comparison to the left graph without induction. Overall growth of the cells at 0 mM Cu(II) concentrations has decreased by 47% after 300 minutes. This effect is a consequence of the burden of expressing genes with a high throughput because of the strong T7 promoter. When growing in copper-containing medium there is also an increasing effect of further growth inhibition visible. The effect can be observed best at a concentration of 2 mM copper (see figure 1). The optical density does not only increase at a reduced rate, it even decreases after approximately 220 minutes. This indicates cell death. Both growth inhibitions can not be observed with <i>E. coli</i> carrying pSB1C3.</article>
  
  

Revision as of 18:06, 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
The growth of the T7 RBS strains expressing either copC, copD, oprC or hmtAafter induction with 0.1 % rhamnose and 0.1 mM IPTG in comparison with non-induced cells is reduced at all tested Cu(II) concentrations in the medium.

Figure 1 shows the growth of E. coli DH5a with BBa_K2638201 (oprC). The right graph shows the growth after induction in comparison to the left graph without induction. Overall growth of the cells at 0 mM Cu(II) concentrations has decreased by 47% after 300 minutes. This effect is a consequence of the burden of expressing genes with a high throughput because of the strong T7 promoter. When growing in copper-containing medium there is also an increasing effect of further growth inhibition visible. The effect can be observed best at a concentration of 2 mM copper (see figure 1). The optical density does not only increase at a reduced rate, it even decreases after approximately 220 minutes. This indicates cell death. Both growth inhibitions can not be observed with E. coli carrying pSB1C3.
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