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

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Accumulation Results

Short Summary

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 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 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 (BBa_K2638002, BBa_K2638004 and BBa_K2638006)

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. coli*KRX 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 (BBa_K2638200, BBa_K2638201 and BBa_K2638204)

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. coli*KRX 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. coli*KRX 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 (BBa_K2638001, BBa_K2638003 and BBa_K2638005)

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. coli*KRX 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. coli*KRX 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 (BBa_K2638000 and BBa_K2638016)

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. coli*KRX strains with BBa_K2638016 (*hmtA* containing plasmid with T7 promoter control, red curve) and as reference a strain with an empty pSB1C3 in *E. coli*KRX 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®).

Discussion

In general our data shows that our copper uptake Biobricks work as expected. The import of copper can be shown by increasing toxic effects on cells, as the growth curves indicate an obvious growth inhibition for OprC and CopD. The CopD encoding Biobricks BBa_K2638002, BBa_K2638004 and BBa_K2638006 show the most impressive results both in the toxicity test and the NPN assay. This result was expected, as the import of Cu(II) occurs actively across the inner cell membrane into the cytoplasm (Lawton et al., 2016), where toxic effects have the strongest impact. The OprC encoding Biobricks BBa_K263200, BBa_K2638201 and BBa_K2638204 also show clear results, as it is a passive importer into the periplasm (Yoneyama & Nakae, 2001).

An interesting observation, the composite Biobrick of CopD with pBAD7araC promoter and RBS BBa_K2638006 shows a higher impact from copper ions during the toxicity tests and a higher fluorescence emission than the T7 promoter BBa_K2638004 and all other copper transport constructs. In this collection this behavior is out of norm and different results were assumed because of the higher promoter strengths of T7 (Balzer et al., 2013). A possible reason for that could be that the chaperone concentration and the concentration of proteins for the buildup of membrane proteins in E. coli KRX just cannot keep up to the expressing level of BBa_K2638004 (Bukau et al., 1990, Gräslund et al., 2008). The other membrane proteins which we equipped with T7 promoters, BBa_K2638204 and BBa_K2638016, have an about 1000 bp longer coding sequence. Therefore, protein expression takes longer for those two constructs and the cellular circumstances are better fitted for their level of protein expression. The missing increase of absorption during the ONPG assay reinforces the assumption that CopD is located in the inner membrane and is specific to metal ions.

HmtA (BBa_K2638000), which fulfills in particular the same function as CopD, shows in the growth curve (Figure 7) as composite Part BBa_K2638016 a very weak toxicity effect, but because of long lasting cloning complications we had no time to repeat that experiment. Nevertheless, it shows that copper ions have a toxic effect on the cell. The ONPG assay shows that HmtA is probably located in the inner membrane and is specific to metal ions as mentioned in the literature (Lewinson et al., 2009).

The NPN assays of CopD and HmtA show positive signals which does not accord to the literature. The NPN assay affects only outer membrane proteins (Helander, 2000). A further experiment is needed by analyzing CopD and HmtA with GFP marker and repeating the NPN assays. If the results are reproducible and the Biobricks are correctly located two possibilities are open: 1. the NPN assay would also work for inner membrane proteins or 2. both proteins have regulatory functions which results in the expression of further outer membrane transporter.

OprC (BBa_K2638200) expression shows lower effects on the dying behavior under increased copper concentrations than copD (BBa_K2638002) and hmtA (BBa_K2638000). This could be due to the fact that OprC is an outer membrane transporter (Yoneyama & Nakae, 1996) and the copper is only transported into the periplasm, where it has lower toxic effects on the cell. The expression of BBa_K2638201 shows a bigger toxicity effect and a higher fluorescence emission than BBa_K2638204 as expected for a T7 promoter. The NPN assay shows a higher membrane permeability that is not higher than the signal that CopD-cultures are emitting which is rather concerning in face of the fact that OprC is an outer membrane protein. The NPN assay should show a higher permeability in this case. The protein probably did not expressed as good as CopD. The ONPG assay showed that OprC is not just an unspecific, unselective porin but a specific transporter for certain metals in the outer cell membrane.

CopC shows the most confusing results. Copper toxicity is nearly not measurable and even if it is just a mediator of copper in the periplasm and not a membrane protein it indicates a higher permeability in the NPN assay. This leaves room for interpretation that CopC either has a regulatory function for expression of membrane proteins or it changes the condition of the membrane to a higher permeability. The ONPG assay shows no signal so it seems to be a periplasmatic mediator protein. One probable function of the periplasmic soluble protein is to deliver Cu(II) to CopD and hence increase its effectivity in copper uptake (Lawton et al., 2016). As CopC and CopD are found ubiquitous in a huge amount of unrelated species (Lawton et al., 2016), the presumption is close that also E. coli expresses a copD-like gene. The associated protein may interact with our introduced CopC and could be supported in its activity. To verify this thesis bioinformatic approaches could help identify such a gene in E. coli and repeat the experiments in knock-out mutants of those genes. Another possible explanation may be a regulatory virtue of CopC.

Further interesting experiments would be the combination of OprC (BBa_K2638200), CopC (BBa_K2638001) with CopD (BBa_2638002) in one biobrick. The combination of an importer into the periplasm and into the cytoplasm could improve the effectivity of uptake dramatically. To further support CopD, CopC could deliver Cu(II) to the transporter through the periplasm. The already performed growth experiments could be performed and measurements of intracellular copper may be conducted.

Balzer, S., Kucharova, V., Megerle, J., Lale, R., Brautaset, T., & Valla, S. (2013).. A comparative analysis of the properties of regulated promoter systems commonly used for recombinant gene expression in Escherichia coli. Microbial cell factories, 12(1), 26.
Bukau, B., & Walker, G. C. (1990).. Mutations altering heat shock specific subunit of RNA polymerase suppress major cellular defects of E. coli mutants lacking the DnaK chaperone. The EMBO journal, 9(12), 4027-4036.
Gräslund, S., Nordlund, P., Weigelt, J., Hallberg, B. M., Bray, J., Gileadi, O., ... & Ming, J. (2008).. Protein production and purification. Nature methods, 5(2), 135.
Helander, I. M., & Mattila-Sandholm, T. (2000). Fluorometric assessment of Gram-negative bacterial permeabilization. Journal of applied microbiology, 88(2), 213-219.
Lawton, T. J., Kenney, G. E., Hurley, J. D., & Rosenzweig, A. C. (2016). The CopC family: structural and bioinformatic insights into a diverse group of periplasmic copper binding proteins. Biochemistry, 55(15).
Lewinson, O., Lee, A. T., & Rees, D. C. (2009). A P-type ATPase importer that discriminates between essential and toxic transition metals. Proceedings of the National Academy of Sciences, 106(12).
Yoneyama, H., & Nakae, T. (1996). Protein C (OprC) of the outer membrane of Pseudomonas aeruginosa is a copper-regulated channel protein. Microbiology, 142(8), 2137-2144.

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 <article>To test whether our accumulation system with the importers <i>oprC</i>, <i>hmtA</i>, <i>copC</i> and <i>copD</i> 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.</article>
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 <article>In general our data shows that our copper uptake Biobricks work as expected. The import of copper can be shown by increasing toxic effects on cells, as the growth curves indicate an obvious growth inhibition for OprC and CopD.  The CopD encoding Biobricks BBa_K2638002, BBa_K2638004 and BBa_K2638006 show the most impressive results both in the toxicity test and the NPN assay. This result was expected, as the import of Cu(II) occurs actively across the inner cell membrane into the cytoplasm (Lawton <i>et al.</i>, 2016), where toxic effects have the strongest impact. The OprC encoding Biobricks BBa_K263200, BBa_K2638201 and BBa_K2638204 also show clear results, as it is a passive importer into the periplasm (Yoneyama & Nakae, 2001).