Team:Bielefeld-CeBiTec/Accumulation Results

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. We also conducted an experiment on the specifity of the ion uptake.

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 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 hmtA 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.

Figure 1 shows the growth of E. coli KRX 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 KRX 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 KRX 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 KRX 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 KRX BBa_K26380016 at different CuSO4 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 KRX 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 DH5α 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 2: 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

CopD is an active copper transport protein in the inner cell membrane. In both strains which expressed the composite parts BBa_2638004 and BBa_K2638006 a higher increase in fluorescence than the pSB1C3 control strain (yellow curve figure 8) was measurable. The strain expressing copD under control of the pBAD/araC promoter with RBS (BBa_K2638006, blue curve in figure 8) showed a maximum of 94.16 ± 10.89 % in the fluorescence emission at 408 nm. This is an increase of 59.03 % to the empty control vector pSB1C3. The copD strain with the T7 promoter (BBa_2638004, red curve in figure 8) showed a maximum regarding the fluorescence of 68.10 ± 2.84 % at 408 nm. This is a increase of 32.99 % to the empty control vector. The difference in the fluorescence mximum at 408 nm of both copD expressing strains compared to the empty vector control show a substantial increase of the membrane permeability of E. coli.
Figure 8: Fluorescence spectra from 382 nm to 454 nm of E. coliKRX strains with BBa_K2638004 (copD containing plasmid with T7 promoter control, red curve), BBa_K2638006 (copD containing plasmid with pBAD/araC promoter with RBS control, blue curve) and as reference a strain with an empty pSB1C3 in a KRX strain (yellow curve). The excitation wavelenght is 355 nm. The OD 600 of cells was adjusted with the NPN stock solution to the same value. The measurement was performed with the Infinite® 200 PRO in a 24 wellplate with flat bottom (Greiner®).
OprC is a copper transport protein in the outer membrane. In both strains which expressed the composite parts BBa_2638201 and BBa_K2638204 a higher increase in fluorescence than the pSB1C3 controll strain (yellow curve figure 9) was measurable. The the strain expressing oprC under control of the pBAD/araC promoter with RBS (BBa_K2638201, blue curve in figure 9) showed a maximum of 71.23 ± 7.78 % in the fluorescence emission at 408 nm. This is a increase of 36.12 % in comparison to the empty control vector. The oprC strain with the T7 promoter (BBa_2638004, red curve in figure 9) showed a maximum regarding the fluorescence of 57.41 % ± 9.52 % at 408 nm. This is an increase of 22.29 % to the empty control vector. Due to the difference in the fluorescence maximum at 408 nm of both oprC expressing strains compared to the empty vector control show a substantial increase of the membrane permeability of E. coli. The difference of fluorescence increase of both oprC Biobricks is substantial. However, the difference in membrane permeability to the pSB1C3 strain and the difference between the both oprC variants was less clear. The BBa_2638201 expressing strain shows a slightly higher permeability than BBa_K2638204 as seen at 406 or 416 nm.
Figure 9: Fluorescence spectra from 382 nm to 454 nm of E. coliKRX strains with BBa_K2638201 (oprC containing plasmid with T7 promoter control, red curve), BBa_K2638204 (oprC containing plasmid with pBAD/araC promoter with RBS control, blue curve) and as reference a strain with an empty pSB1C3 in E. coliKRX strain (yellow curve). The excitation wavelength is 355 nm. The OD 600 of cells was adjusted with the NPN stock solution to the same value. The measurement was performed with the Infinite® 200 PRO in a 24 wellplate with flat bottom (Greiner®).
CopC is a copper mediator protein which is localized in the periplasm. In both strains which expressed the composite parts BBa_2638003 and BBa_K2638005 a higher increase in fluorescence than the pSB1C3 control strain (yellow curve, figure 10) was measurable. The copC strain with the T7 promoter (BBa_2638003, red curve in figure 10) showed a maximum regarding the fluorescence of 75.23 % ± 17.55 % at 408 nm. This is an increase of 40.20 % to the empty control vector. The the strain expressing copC under control of the pBAD/araC promoter with RBS (BBa_K2638003, blue curve in figure 10) showed a maximum of 51.42 ± 10.59 % of fluorescence emission at 408 nm. This is a increase of 16.30 % to the empty control vector. A slight difference of the fluorescence emission of both copC expressing strains in comparison to the pSB1C3 empty vector control strain can be observed despite the big error bars. However, the difference between the both copC variants could not be postulated definitely.
Figure 10: Fluorescence spectra from 382 nm to 454 nm of E. coliKRX strains with BBa_K2638003 (copC containing plasmid with T7 promoter control, red curve), BBa_K2638005 (copC containing plasmid with pBAD/araC promoter with RBS control, blue curve) and as reference a strain with an empty pSB1C3 in E. coliKRX strain (yellow curve). The excitation wavelength is 355 nm. The OD 600 of cells was adjusted with the NPN stock solution to the same value. The measurement was performed with the Infinite® 200 PRO in a 24 wellplate with flat bottom (Greiner®).
HmtA is a copper and zinc specific transporter in the inner cell membrane. In the strain which expressed the composite part BBa_2638016 an increase in fluorescence compared to the pSB1C3 control strain (blue curve, figure 11) was measurable. The hmtA strain with the T7 promoter (BBa_2638016, red curve in figure 11) showed a maximum regarding the fluorescence of 62.35 ± 4.47 % at 408 nm. This is an increase of 27.23 % to the empty control vector.
Figure 11: Fluorescence spectra from 382 nm to 454 nm of E. coliKRX strains with BBa_K2638016 (hmtA containing plasmid with T7 promoter control, red curve) and as reference a strain with an empty pSB1C3 in E. coliKRX strain (blue curve). The excitation wavelength is 355 nm. The OD 600 of cells was adjusted with the NPN stock solution to the same value. The measurement was performed with the Infinite® 200 PRO in a 24 wellplate with flat bottom (Greiner®).

Specific Uptake Assay

An orhto-Nitrophenyl-β-galactoside (ONPG) assay was performed with eight different pSB1C3 constructs to show that our copper transport Biobricks do not unspecifically take up different substrates.

The ONPG assay shows no unspecific uptake for all eight analyzed cultures (figure 12). As a reference an E. coli KRX with the empty vector pSB1C3 has been used. Over a period of t = 3000 s no increase of absorption (λ = 400 nm) could be detected.
Figure 12: Results of the ONPG assay. The absorption spectra are measured at λ = 400 nm over t = 3000 s. The measurement was performed with the Infinite® 200 PRO in a 24 wellplate with flat bottom (Greiner®).
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