Difference between revisions of "Part:BBa K1916118"

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]

@@ Line 3: / Line 3: @@
 <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. Cells did not appear to express photoactive proteins, suggesting that the protein requires a different organic co-factor other than phycocyanobilin.
+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).
 [[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. There does not appear to be any peak absorbance.]]
+[[Image:K1916118_cbcr8_spectra.jpeg|600px|thumb|left|Fig.2 Absorbance spectra of the CBCR8 protein extracted from the cells.]]