Difference between revisions of "Part:BBa K1916103"
 
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<partinfo>BBa_K1916103 short</partinfo>
 
<partinfo>BBa_K1916103 short</partinfo>
  
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A GAF domain of the cyanobacteriochrome (CBCR) Cha6605_4344 with an intein/chitin-binding domain fused to the C-terminus for purification. Expression regulated by a pBAD promoter. As with most CBCRs, NpF requires a phycocyanobilin co-factor, and must therefore be expressed in a system with heme oxygenase (BBa_I15008) and PcyA (BBa_I15009). Cells appear to be off-yellow under normal light, and may be difficult to distinguish from a negative control.
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A GAF domain of the cyanobacteriochrome (CBCR) Cha6605_4344 with an intein/chitin-binding domain fused to the C-terminus for purification. Expression regulated by a pBAD promoter. Cells did not appear to express photoactive proteins, suggesting that the protein requires a different organic co-factor other than phycocyanobilin.
  
 
==Characterization==
 
==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 with increasing levels of arabinose to study the vibrancy of the cells once harvested (Fig.1).
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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:K1916103_cha_induced_vs_uninduced.jpeg|500px|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:K1916103_cha_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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Latest revision as of 06:54, 27 October 2016


Ara-Inducible Cha6605_4344g2 Expression System

A GAF domain of the cyanobacteriochrome (CBCR) Cha6605_4344 with an intein/chitin-binding domain fused to the C-terminus for purification. Expression regulated by a pBAD promoter. Cells did not appear to express photoactive proteins, suggesting that the protein requires a different organic co-factor other than phycocyanobilin.

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 Comparison of pellets from a negative control strain (left) and from cells spun down 4 hrs after being induced (right).

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 740
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1260
    Illegal AgeI site found at 1350
  • 1000
    COMPATIBLE WITH RFC[1000]