Difference between revisions of "Part:BBa K1916118"
 
(3 intermediate revisions by the same user not shown)
Line 3: Line 3:
 
<partinfo>BBa_K1916118 short</partinfo>
 
<partinfo>BBa_K1916118 short</partinfo>
  
−
A GAF domain of a cyanobacteriochrome (CBCR) designated CBCR8, found through metagenomic mining and predicted to appear yellow by the 2016 Davis team's CBCR color predictive program, with an intein/chitin-binding domain fused to the C-terminus for purification. Expression regulated by a pBAD promoter. CBCR8 possibly requires a phycocyanobilin co-factor, and must be expressed in a system with heme oxygenase (BBa_I15008) and PcyA (BBa_I15009). CBCR8 cells appear yellow under normal light, and may be difficult to distinguish from a negative control.
+
A GAF domain of a cyanobacteriochrome (CBCR) designated CBCR8, found through metagenomic mining and predicted to appear yellow by the 2016 Davis team's CBCR color predictive program, with an intein/chitin-binding domain fused to the C-terminus for purification. Expression regulated by a pBAD promoter. As with most CBCRs, CBCR8 requires a phycocyanobilin co-factor, and must therefore be expressed in a system with heme oxygenase (BBa_I15008) and PcyA (BBa_I15009). CBCR8 expressing cells appear yellow under normal light, which may make it difficult to differentiate from negative controls.
  
 
==Characterization==
 
==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).
 
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).
 
[[Image:K1916118_cbcr8_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).]]
 
[[Image:K1916118_cbcr8_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).]]
 +
[[Image:K1916118_cbcr8_spectra.jpeg|600px|thumb|left|Fig.2 Absorbance spectra of the CBCR8 protein extracted from the cells.]]
 +
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 02:31, 28 October 2016


Ara-Inducible CBCR8 Expression System

A GAF domain of a cyanobacteriochrome (CBCR) designated CBCR8, found through metagenomic mining and predicted to appear yellow by the 2016 Davis team's CBCR color predictive program, with an intein/chitin-binding domain fused to the C-terminus for purification. Expression regulated by a pBAD promoter. As with most CBCRs, CBCR8 requires a phycocyanobilin co-factor, and must therefore be expressed in a system with heme oxygenase (BBa_I15008) and PcyA (BBa_I15009). CBCR8 expressing cells appear yellow under normal light, which may make it difficult to differentiate from negative controls.

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).
Fig.2 Absorbance spectra of the CBCR8 protein extracted from the cells.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 131
    Illegal SpeI site found at 743
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
    Illegal SpeI site found at 743
  • 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 743
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 131
    Illegal SpeI site found at 743
    Illegal NgoMIV site found at 1239
    Illegal AgeI site found at 1329
  • 1000
    COMPATIBLE WITH RFC[1000]