Difference between revisions of "Part:BBa K1916118"
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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:K1916118_cbcr8_induced_vs_uninduced.jpeg|400px|thumb|left|Fig.1 Cells expressing | + | [[Image:K1916118_cbcr8_induced_vs_uninduced.jpeg|400px|thumb|left|Fig.1 Cells expressing CBCR8 spun down 4 hrs after induction.]] |
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Revision as of 08:02, 25 October 2016
Ara-Inducible CBCR8 Expression System
A GAF domain of a cyanobacteriochrome (CBCR) designated CBCR8, found through metagenomic mining and predicted to appear yellow 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. CBCR8 possibly requires a phycocyanobilin co-factor, and must be expressed in a system with heme oxygenase (BBa_I15008) and PcyA (BBa_I15009). CBCR8 cells appear off-yellow under normal light, and may be difficult to distinguish from a negative control.
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 743 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 125
Illegal SpeI site found at 743 - 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 743 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 131
Illegal SpeI site found at 743
Illegal NgoMIV site found at 1239
Illegal AgeI site found at 1329 - 1000COMPATIBLE WITH RFC[1000]