Difference between revisions of "Part:BBa K1916116"

 
 
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<partinfo>BBa_K1916116 short</partinfo>
 
<partinfo>BBa_K1916116 short</partinfo>
  
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The CBCR6 protein (see BBa_K1916016) under control of a strong pBAD promoter.  
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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.
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==Characterization==
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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).
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[[Image:K1916116_cbcr6_pellet.jpeg|200px|thumb|left|Fig.1 Cells expressing CBCR6 spun down 4 hrs after induction.]]
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[[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).

Fig.1 Cells expressing CBCR6 spun down 4 hrs after induction.
Fig.2 Absorbance spectra of the CBCR6 protein extracted from the cells. Peak absorbance occurs around 640nm.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 131
    Illegal SpeI site found at 692
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
    Illegal SpeI site found at 692
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 131
    Illegal SpeI site found at 692
  • 25
    INCOMPATIBLE 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
  • 1000
    COMPATIBLE WITH RFC[1000]