Difference between revisions of "Part:BBa K1916105"

Revision as of 07:33, 25 October 2016

Ara-Inducible Ga0100955_11928g3 Expression System

A GAF domain of the cyanobacteriochrome (CBCR) Ga0100955_11928 with an intein/chitin-binding domain fused to the C-terminus for purification. Expression regulated by a pBAD promoter. As with most CBCRs, Ga_g3 requires a phycocyanobilin co-factor, and must therefore be expressed in a system with heme oxygenase (BBa_I15008) and PcyA (BBa_I15009). Ga0100955_11928g3 appear to be off-yellow under normal light, and may be difficult to distinguish from a negative control. RFC10 compatible.

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, 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).

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 744
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1264
Illegal AgeI site found at 1354
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K1916105 short</partinfo>
-The Ga0100955_11928g3 protein (see BBa_K1916005) under control of a strong pBAD promoter. RFC10 compatible.
+A GAF domain of the cyanobacteriochrome (CBCR) Ga0100955_11928 with an intein/chitin-binding domain fused to the C-terminus for purification. Expression regulated by a pBAD promoter. As with most CBCRs, Ga_g3 requires a phycocyanobilin co-factor, and must therefore be expressed in a system with heme oxygenase (BBa_I15008) and PcyA (BBa_I15009). Ga0100955_11928g3 appear to be off-yellow under normal light, and may be difficult to distinguish from a negative control. RFC10 compatible.
+==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, cultures were induced to a final culture concentration of 1mM arabinose to study the color of the cells once harvested (Fig.1).
+[[Image:K1916105_g3_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).]]
 <!-- Add more about the biology of this part here