Part:BBa_K4259005
copA from E. coli
The copA gene is a copper-translocating P-type ATPase.
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 copA gene has been shown to be involved in copper resistance. However, it has also been shown that copA is induced by the addition of silver. Thus, we attempted to check if the protein functions to reduce silver and gold ions in E.coli.
Design We obtained the copA 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 CopA.
Characterization We set up an experiment to test CopA’s ability to form bimetallic nanoparticles in a cell-free system. In this set up we grew the strains in Müller Hinten broth (MH broth) and either lysed the cells or spun them down and used the supernatant for the production of nanoparticles. The copA 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. 1. Absorbance graph obtained at 800 nm after addition of gold and silver ions to Mueller Hinton (MH) broth media, BL21 supernatant, MH broth with CopA lysate and a combination of CopA lysate with BL21 supernatant. These readings were taken at 24h after the addition of gold and silver salts.
The graph shows absorbance obtained for CopA at 800 nm. High absorbance was observed 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 only the medium with lysate and silver and gold. Nonetheless, this shows that CopA 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.
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