Team:Bielefeld-CeBiTec/Basic Part

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Basic Parts

Short Summary

Our best new basic part is BBa_K2638002, copD, an inner membrane importer for copper ions. It is part of our accumulation system in E. coli. In its native organism Pseudomonas brassicacearum 3RE2-7 it is expressed next to copC, which supports effective uptake. Also we have listed all our basic parts on this page.

Best New Basic Part

We expressed the copD gene in two different expression systems, one with a T7 promoter and RBS (BBa_K2638004) and the other with a pBAD/araC and RBS system (BBa_K2638006). All results also in comparison with the other importers we characterized can be found on our accumulation results page.

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.

Figure 2: Growth curves of E. coli DH5α BBa_K2638006 at different CuSO₄ concentrations. Left: No induction. Right: Induction started simultaneously with inoculation with 1 % arabinose.

Using the growth curve data from E. coliDH5α carrying BBa_K2638006 we also compared relative growth of a strain at 2 mM or 3 mM Cu(II) against the growth at 0 mM Cu(II) for not induced and induced cultures (figure 3). 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 3: 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. Afterwards, fluorescence was measured in the range from 380 – 550 nm after exitation with light with the wavelength of 355 nm. The fluorescence values for every measurement were normalized to the fluorescence value at 382 nm.

In both strains which expressed the copD composite parts BBa_2638004 (T7 promoter, RBS and copD) and BBa_K2638006 (pBAD/araC promoter, RBS and copD) a higher increase in the percental fluorescence increase was shown compared to the pSB1C3 control strain (yellow curve figure 3). The strain expressing copD under control of the pBAD/araC promoter with RBS (BBa_K2638006, blue curve in figure 3) 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 3) showed a maximum regarding the fluorescence of 68.10 ± 2.84 % at 408 nm. This is an increase of 32.99 % in comparison to the empty vector control. The difference increased fluorescence at 408 nm of both copD expressing strains compared to the empty vector control show a substantial increased of the membrane permeability of E. coli. This also indicates substantial uptake.

Figure 3: Relative fluorescence increase 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 an empty vector control an empty pSB1C3 in E. coli KRX (yellow curve). The excitation wavelenght is 355 nm. The OD₆₀₀ 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®).

Specifity of Cu(II) uptake is shown with an ONPG-assay (orhto-Nitrophenyl-β-galactoside assay). The ONPG assay shows no unspecific uptake for copD and the other analyzed constructs (figure 4).

Figure 4: 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®).

For a complete discussion look up the results page

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.

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.

Collection of Basic Parts

For our metal resource recovery system we build lots of new biobricks for accumulation and storage of metal ions, toxicity counteractions and nanoparticle formation. We also designed parts for our siRNA expression and analyzation tool to regulate expression of individual genes on RNA level.

Table 1:All BioBricks submitted by iGEM Bielefeld CeBiTec 2018.

Identifier	Type	Description	Designer	Length
BBa_K2638000	Coding	HmtA	Erika Schneider	1920
BBa_K2638001	Coding	CopC coding sequence	Erika Schneider	387
BBa_K2638002	Coding	CopD CDS	Erika Schneider	912
BBa_K2638000	Coding	Heavy Metal Transporter (hmtA)	Erika Schneider	1920
BBa_K2638100	Coding	alkyl hydroperoxide reductase, C22 subunit (ahpC)	Johannes Ruhnau	564
BBa_K2638103	Coding	glutathione synthetase (gshB)	Johannes Ruhnau	951
BBa_K2638105	Coding	catalase HPI (katG)	Johannes Ruhnau	2181
BBa_K2638106	Coding	superoxide dismutase (sodA)	Johannes Ruhnau	621
BBa_K2638120	Coding	glutathione peroxidase (btuE)	Johannes Ruhnau	552
BBa_K2638121	Coding	glutathione reductase (gor)	Johannes Ruhnau	1353
BBa_K2638150	Coding	Glutathione gamma-glutamylcysteinyltransferase 1	Matthias Otto	1458
BBa_K2638151	Coding	Glutathione gamma-glutamylcysteinyltransferase 1 with RBS and pTet promoter	Matthias Otto	1535
BBa_K2638152	Coding	Glutathione gamma-glutamylcysteinyltransferase 1 with RBS and Anderson promoter	Matthias Otto	1519
BBa_K2638153	Coding	Glutathione gamma-glutamylcysteinyltransferase 1 with AraBAD promoter and RBS	Matthias Otto	2694
BBa_K2638200	Coding	OprC (TonB dependent copper transport porin)	Jakob Zubek	2127
BBa_K2638426	Reporter	mRFP+	Levin Joe Klages	827
BBa_K2638601	Coding	Human Ferritin Heavy Chain Copper Enhanced Binding	Christoph Geske	552
BBa_K2638602	Coding	Human Ferritin Heavy Chain E-Helix Removed Weaker Binding	Christoph Geske	480
BBa_K2638603	Coding	Human Ferritin Heavy Chain Copper Enhanced Binding and E-Helix removed	Christoph Geske	474
BBa_K2638701	Generator	Seamless siRNA Expression Vektor pGuide	Antonin Lenzen	1780
BBa_K2638702	Generator	Seamless siRNA Expression Vektor for Hfq binding siRNAs	Antonin Lenzen	1865
BBa_K2638707	Reporter	siRNA Target Vector with BFP and a GGGGS Linker	Antonin Lenzen	2779
BBa_K2638708	Reporter	siRNA Target Vector with BFP and an EAAAK Linker	Antonin Lenzen	2779
BBa_K2638709	Reporter	siRNA Target Vector with BFP and a cMyc Linker	Antonin Lenzen	2779
BBa_K2638710	Reporter	siRNA Target Vector with BFP and XP Linker	Antonin Lenzen	2779
BBa_K2638711	Reporter	siRNA Target Vector with AmilCP and a GGGGS Linker	Antonin Lenzen	1226
BBa_K2638712	Reporter	siRNA Target Vector with AmilCP and EAAAK Linker	Antonin Lenzen	1226
BBa_K2638713	Reporter	siRNA Target Vector with AmilCP and a cMyc Linker	Antonin Lenzen	1226
BBa_K2638714	Reporter	siRNA Target Vector with AmilCP and XP Linker	Antonin Lenzen	1226
BBa_K2638751	Regulatory	Synthetic Origin of Replication B	Antonin Lenzen	837
BBa_K2638752	Regulatory	pLac including a TetO sequence in place of the CAP binding sequence	Antonin Lenzen	206
BBa_K2638753	Regulatory	pLac including a Tet operator sequence behind the Lac operator	Antonin Lenzen	213
BBa_K2638754	Coding	E. coli Bacterioferritin Bfr	Antonin Lenzen	477
BBa_K2638755	Coding	Ferredoxin reductase	Antonin Lenzen	747
BBa_K2638756	Coding	Bacterial non-heme ferritin FtnA from Escherichia coli	Antonin Lenzen	498
BBa_K2638757	Coding	Ferric Uptake Regulator (Fur) from Escherichia coli K12	Antonin Lenzen	447
BBa_K2638758	Generator	Seamless siRNA Expression Vektor for Omp-Utr protected siRNAs	Antonin Lenzen	1897
BBa_K2638759	Generator	Seamless siRNA Expression Vektor with an Hfq binding tag and OmpA 5'-UTR	Antonin Lenzen	1982
BBa_K2638998	Coding	araBAD and RBS and Mutated Human Ferritin Heavy Chain	Vanessa Krämer	1566
BBa_K2638999	Coding	Mutated Human Ferritin Heavy Chain	Vanessa Krämer	552

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References ▾

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).