Part:BBa_K4259004
cueO from E. coli
CueO is a multicopper oxidase involved in copper tolerance.
Usage and Biology
Binanox aimed to synthesize bimetallic nanoparticles with a silver core and golden spikes by overexpressing certain genes in E. coli by using a cell-free system. The cueO gene is involved in copper detoxification through efflux and by oxidizing Cu+ to Cu2+. It is also upregulated in many heavy metal stress situations.1 CueO in E. coli is the equivalent of CopA in Cupriavidus metallidurans (BBa_K4259003), which is another protein we used during the Binanox project. Previous studies have shown that upon exposure to gold ions copA is over-expressed in C. metallidurans, thus we attempted to check if the protein also functions to reduce silver and gold ions in E.coli.
Design
We obtained the cueO gene from the ASKA collection, where the gene is in the plasmid pCA24N. This plasmid was then transformed into E. coli BL21. The strains were induced with IPTG to express cueO.
Characterization
The strain was induced with IPTG to express CueO, this was shown on an SDS gel. The protein size for melA is 57kDA.
Fig. 1. SDS gel comparing non-induced CueO to induced CueO. 3. CueO non-induced 4. CueO induced with IPTG 5. protein ladder.
We set up an experiment to test CueO’s ability to form bimetallic nanoparticles in a cell-free system. In this set up we grew the strains in MH broth and either lysed the cells or spun them down and used the supernatant for the production of nanoparticles. The cueO strains were compared to the control of E. coli BL21 strains (WT). The silver and gold was added under the form of AgNO3 and HAuCl4 salts. The absorbance was measured after 24 hours.
Fig. 2. Absorbance graph obtained at 800 nm after addition of gold and silver ions to Mueller Hinton (MH) broth media, BL21 supernatant, MH-broth with CueO lysate and a combination of CueO lysate and BL21 supernatant. These readings were taken at 24h after the addition of gold and silver salts.
The graph shows absorbance obtained for CueO at 800 nm. As can be observed in the graph, the highest recorded absorbance is for the samples containing medium with salts. This can be attributed to the presence of tryptone in the medium which acts as a strong reducing agent. However, upon the addition of lysate to supernatant with gold and silver salts, the absorbance value drops. A low absorbance is also recorded for lysate with silver and gold. Nonetheless, this shows that CueO is capable of forming nanoparticles, but that maybe lysing the cells is not the most optimal method for a cell-free system. You can find more information on this on the wiki page from the Binanox team.
References
1. Singh, S. K., Grass, G., Rensing, C. & Montfort, W. R. Cuprous oxidase activity of CueO from Escherichia coli. J Bacteriol 186, 7815–7817 (2004).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 238
Illegal PstI site found at 1051 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 238
Illegal PstI site found at 1051 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1065
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 238
Illegal PstI site found at 1051 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 238
Illegal PstI site found at 1051
Illegal NgoMIV site found at 399
Illegal NgoMIV site found at 1011
Illegal NgoMIV site found at 1383
Illegal AgeI site found at 798 - 1000COMPATIBLE WITH RFC[1000]
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