Difference between revisions of "Part:BBa K1916102"

Latest revision as of 06:48, 27 October 2016

Ara-Inducible Cyan7427_1390g3 Expression System

A GAF domain of the cyanobacteriochrome (CBCR) Cyan7427_1390 with an intein/chitin-binding domain fused to the C-terminus for purification. Expression regulated by a pBAD promoter. As with most CBCRs, Cyan7427 requires a phycocyanobilin co-factor, and must therefore be expressed in a system with heme oxygenase (BBa_I15008) and PcyA (BBa_I15009). Cyan7427_1390g3 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 Cyan7427, spun down 4 hrs after being induced by different concentrations of arabinose.

Fig.2 Absorbance spectra of the Cyan protein extracted from the cells. Peak absorbance occurs around 695nm.

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 731
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1251
Illegal AgeI site found at 1341
1000
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K1916102 short</partinfo>
-A GAF domain of the cyanobacteriochrome (CBCR) Cyan7427_1390 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). Cyan7427_1390g3 expresses well in E. coli and cells appear vibrant blue-green under normal light.
+A GAF domain of the cyanobacteriochrome (CBCR) Cyan7427_1390 with an intein/chitin-binding domain fused to the C-terminus for purification. Expression regulated by a pBAD promoter. As with most CBCRs, Cyan7427 requires a phycocyanobilin co-factor, and must therefore be expressed in a system with heme oxygenase (BBa_I15008) and PcyA (BBa_I15009). Cyan7427_1390g3 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.
+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).
 [[Image:K1916102_cyan_induction.jpeg|600px|thumb|left|Fig.1 Pellets of cells expressing Cyan7427, spun down 4 hrs after being induced by different concentrations of arabinose.]]
+[[Image:K1916102_cyan_spectra.jpeg|600px|thumb|left|Fig.2 Absorbance spectra of the Cyan protein extracted from the cells. Peak absorbance occurs around 695nm.]]