Difference between revisions of "Part:BBa K1916102"

 
 
(5 intermediate revisions by the same user not shown)
Line 3: Line 3:
 
<partinfo>BBa_K1916102 short</partinfo>
 
<partinfo>BBa_K1916102 short</partinfo>
  
−
The Cyan7427_1390g3 protein (see BBa_K1916001) under control of a strong pBAD promoter.  
+
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).
 +
[[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.]]
 +
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

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]