Difference between revisions of "Part:BBa K1916100"
 
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A GAF domain of the cyanobacteriochrome (CBCR) NpF2164 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). NpF2164g5 expresses well in E. coli and cells appear vibrant blue-green under normal light.
 
A GAF domain of the cyanobacteriochrome (CBCR) NpF2164 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). NpF2164g5 expresses well in E. coli and cells appear vibrant blue-green under normal light.
  
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[[Image:K1916100_npf_induction.jpeg|200px|thumb|left|Pellets of cells expressing NpF, spun down 4 hrs after being induced by different concentrations of arabinose.]]
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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 with increasing levels of arabinose to study the vibrancy of the cells once harvested (Fig.1).
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[[Image:K1916100_npf_induction.jpeg|600px|thumb|left|Fig.1 Pellets of cells expressing NpF, spun down 4 hrs after being induced by different concentrations of arabinose.]]
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[[Image:K1916100_npf_spectra.jpeg|600px|thumb|left|Fig.2 Absorbance spectra of the NpF protein extracted from the cells. Peak absorbance occurs around 640nm.]]
  
 
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Latest revision as of 06:46, 27 October 2016


Ara-Inducible NpF2164g5 Expression System

A GAF domain of the cyanobacteriochrome (CBCR) NpF2164 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). NpF2164g5 expresses well in E. coli and cells appear vibrant blue-green under normal light.

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 with increasing levels of arabinose to study the vibrancy of the cells once harvested (Fig.1).

Fig.1 Pellets of cells expressing NpF, spun down 4 hrs after being induced by different concentrations of arabinose.
Fig.2 Absorbance spectra of the NpF 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 686
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
    Illegal SpeI site found at 686
  • 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 686
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 131
    Illegal SpeI site found at 686
    Illegal NgoMIV site found at 1182
    Illegal AgeI site found at 1272
  • 1000
    COMPATIBLE WITH RFC[1000]