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 assays
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.Figure 1: Growth curves measuring OD 600 with E. coli DH5a BBa_K2638201 at different CuSO4 concentrations. Left: No induction. Right: Induction started simultanously with inoculation with 0.1 % rhamnose and 0.1 mM IPTG.
This could, as in all other experiments, be reproduced starting induction also 30 or 60 minutes before (see figure 2). A decrease of optical density can be observed in either measurement series. Because of different moments of decreasing OD or cell death the results can not be merged to examine mean values.Figure 2: Growth curves measuring OD 600 with E. coli DH5a BBa_K2638201 at different CuSO4 concentrations. Left: Induction with 0.1 % rhamnose and 0.1 mM IPTG 30 minutes before inoculation. Right: Induction with 0.1 % rhamnose and 0.1 mM IPTG 60 minutes before inoculation.
The growth curves for BBa_K2638003 (copC) show an effect for copper concentrations upon induction, which indicates that the gene is expressed (see figure 3). The decrease in OD from toxicity from copper uptake is visible at 2 mM Cu(II) after about 300 minutes.Figure 3: Growth curves measuring OD 600 with E. coli DH5a BBa_K2638003 at different CuSO4 concentrations. Left: No induction. Right: Induction started simultanously with inoculation with 0.1 % rhamnose and 0.1 mM IPTG.
Growth curves of hmtA (BBa_K2638016) only show a very weak effect, but also the expression itself does not alter cell growth much (see figure 4). This experiment should be repeated. Figure 4: Growth curves measuring OD 600 with E. coli DH5a BBa_K26380016 at different CuSO<4/sub> concentrations. Left: No induction. Right: Induction started simultanously with inoculation with 0.1 % rhamnose and 0.1 mM IPTG.
With BBa_K2638004 (copD) the effect of cell death can also be observed. Here gene expression in an environment without supplementary Cu(II) reduces OD 600 by 33% 400 min after growth initiation compared to 47 % for the oprC construct BBa_K2638201 (see figure 5). With copD in combination with pBAD/araC/RBS (BBa_K2638006) this can be reproduced and the effect there is more obvious (see figure 6).Figure 5:Growth curves measuring OD 600 with E. coli DH5a BBa_K2638004 at different CuSO4 concentrations. Left: No induction. Right: Induction started simultanously with inoculation with 0.1 % rhamnose and 0.1 mM IPTG.
Figure 6:Growth curves measuring OD 600 with E. coli DH5a BBa_K2638006 at different CuSO4 concentrations. Left: No induction. Right: Induction started simultanously with inoculation with 1% arabinose.
For the underlying data of BBa_K2638006 we also compared relative growth of a strain at 2 mM and 3 mM Cu(II) against the growth at 0 mM Cu(II) for not induced and induced cultures (see figure 7). 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 7: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. The fluorescence was excited with 355 nm and fluorescence was measured from 380 - 550 nm. The fluorescence values were divided by the fluorescence data at 382 nm. The fluorescence data reached at 382 nm a minimum and are marking that way the starting point of the measurement.The equation (1) was furthermore used to calculate the percent increase of the fluorescence. Thus the increasing of fluorescence could be easier to determine.
The NPN assays showed a higher fluorescence increase for all outer membrane transport systems compared to the strain with the empty vector (pSB1C3) as a control.
Table 1: Parts used in toxicity assay (growth curves)
Biobrick number
contains
Fluorescence at 408 nm
F Error
ΔF to pSB1C3
x axis intersection nm
--
pSB1C3
35.12
7.78
--
431
BBa_K2638201
T7 oprC
71.24
9.52
36.11
443
BBa_K2638204
pBAD/araC RBS oprC
57.41
17.55
22.29
440
BBa_K2638003
T7 copC
75.32
10.59
40.20
447
BBa_K2638005
pBAD/araC RBS copC
51.42
2.85
16.30
441
BBa_K2638004
T7 copD
68.11
10.89
32.98
443
BBa_K2638006
pBAD/araC RBS copD
94.16
4.47
59.04
455
BBa_K2638016
T7 hmtA
62.35
7.13
27.23
443
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