Difference between revisions of "Part:BBa K1916105"

 
 
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<partinfo>BBa_K1916105 short</partinfo>
 
<partinfo>BBa_K1916105 short</partinfo>
  
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The Ga0100955_11928g3 protein (see BBa_K1916001) under control of a strong pBAD promoter. RFC10 compatible.
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A GAF domain of the cyanobacteriochrome (CBCR) Ga0100955_11928 with an intein/chitin-binding domain fused to the C-terminus for purification. Expression regulated by a pBAD promoter. As with most CBCRs, it requires a phycocyanobilin co-factor, and must therefore be expressed in a system with heme oxygenase (BBa_I15008) and PcyA (BBa_I15009). Ga0100955_11928g3 does not express very well in E. coli but the proteins can still be visibly observed in spun down pellets of the cells compared to negative controls. Cells appear somewhat blue-green under normal light. RFC10 compatible.
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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, 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:K1916105_g3_induced_vs_uninduced.jpeg|400px|thumb|left|Fig.1 Comparison of pellets from a negative control strain (left) and from cells spun down 4 hrs after being induced (right).]]
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[[Image:K1916105_g3_spectra.jpeg|600px|thumb|left|Fig.2 Absorbance spectra of the G3 protein extracted from the cells. Peak absorbance occurs around 740nm.]]
  
 
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Latest revision as of 06:51, 27 October 2016


Ara-Inducible Ga0100955_11928g3 Expression System

A GAF domain of the cyanobacteriochrome (CBCR) Ga0100955_11928 with an intein/chitin-binding domain fused to the C-terminus for purification. Expression regulated by a pBAD promoter. As with most CBCRs, it requires a phycocyanobilin co-factor, and must therefore be expressed in a system with heme oxygenase (BBa_I15008) and PcyA (BBa_I15009). Ga0100955_11928g3 does not express very well in E. coli but the proteins can still be visibly observed in spun down pellets of the cells compared to negative controls. Cells appear somewhat blue-green under normal light. RFC10 compatible.

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, cultures were induced to a final culture concentration of 1mM arabinose to study the color of the cells once harvested (Fig.1).

Fig.1 Comparison of pellets from a negative control strain (left) and from cells spun down 4 hrs after being induced (right).
Fig.2 Absorbance spectra of the G3 protein extracted from the cells. Peak absorbance occurs around 740nm.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
    Illegal BamHI site found at 744
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1264
    Illegal AgeI site found at 1354
  • 1000
    COMPATIBLE WITH RFC[1000]