Difference between revisions of "Part:BBa K1916116"
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==Characterization== | ==Characterization== | ||
The ara-inducible system was cloned into pSB1C3 and co-transformed into DH5-alpha cells along with a plasmid containing heme oxygenase and PcyA. After growing the cells to OD=0.9-1 in Terrific Broth, the cultures were induced to a final culture concentration of 1mM arabinose to study the color of the cells once harvested (Fig.1). | The ara-inducible system was cloned into pSB1C3 and co-transformed into DH5-alpha cells along with a plasmid containing heme oxygenase and PcyA. After growing the cells to OD=0.9-1 in Terrific Broth, the cultures were induced to a final culture concentration of 1mM arabinose to study the color of the cells once harvested (Fig.1). | ||
| − | [[Image:K1916116_cbcr6_pellet.jpeg|200px|thumb|left|Fig.1 | + | [[Image:K1916116_cbcr6_pellet.jpeg|200px|thumb|left|Fig.1 Cells expressing CBCR6 spun down 4 hrs after induction.]] |
| + | [[Image:K1916116_cbcr6_spectra.jpeg|600px|thumb|left|Fig.2 Absorbance spectra of the CBCR6 protein extracted from the cells. Peak absorbance occurs around 640nm.]] | ||
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Latest revision as of 06:49, 27 October 2016
Ara-Inducible CBCR6 Expression System
A GAF domain of a cyanobacteriochrome (CBCR) designated CBCR6, found through metagenomic mining and predicted to appear blue by the 2016 Davis team's CBCR color predictive program, with an intein/chitin-binding domain fused to the C-terminus for purification. Expression regulated by a pBAD promoter. It was confirmed after co-transformation and expression that CBCR6 requires a phycocyanobilin co-factor, and must therefore be expressed in a system with heme oxygenase (BBa_I15008) and PcyA (BBa_I15009). It was discovered that CBCR6 cells express very well in E. coli and appear dark blue/purple under normal light, thus confirming the initial program prediction.
Characterization
The ara-inducible system was cloned into pSB1C3 and co-transformed into DH5-alpha cells along with a plasmid containing heme oxygenase and PcyA. After growing the cells to OD=0.9-1 in Terrific Broth, the cultures were induced to a final culture concentration of 1mM arabinose to study the color of the cells once harvested (Fig.1).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 131
Illegal SpeI site found at 692 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 125
Illegal SpeI site found at 692 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 65
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 131
Illegal SpeI site found at 692 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 131
Illegal SpeI site found at 692
Illegal NgoMIV site found at 1188
Illegal AgeI site found at 1278 - 1000COMPATIBLE WITH RFC[1000]