Part:BBa_K497004:Experience

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Applications of BBa_K497004

Subcloned part in pGEM was expressed in BL21(DE3) cells. The cells were grown in 3 ml of LB till the OD600 was 0.5. Protein expression was induced with 1 mM IPTG and biliverdin was added at different concentrations to see if the Bacteriophytochrome would assemble in vivo. The treated cultures were kept at 4 C for 2 days. They were then harvested by centrifugation, washed twice with Tris buffered saline and the cells were lysed in SDS-PAGE loading buffer by heating to 95 C for 5 minutes. The sample was sheared by passage through a 250 uL Hamilton syringe 10 times and centrifuged at 14,000g for 10 minutes. 10 uL of supernatant containing approximately 50 ug of protein was loaded onto a 4-20% SDS-PAGE gel and the gel was subjected to electrophoresis until the blue dye front was at the bottom of the gel.

Normal assembly of Bacteriophytochrome occurs by autocatalytic covalent attachment of biliverdin which can be visualised by Zn2+-induced fluorescence at ~700nm, by incubation with 20 mM zinc acetate for 20 minutes followed by imaging on a Odyssey IR imaging system. The figure attached shows the fluorescence due to Bph with biliverdin attached and the coomassie stained gel after fluorescent imaging. Please see our Notebook3 section on our wiki: http://2010.igem.org/Team:Macquarie_Australia/Notebook3

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