Difference between revisions of "Part:BBa K1916103"

Revision as of 07:17, 25 October 2016

Ara-Inducible Cha6605_4344g2 Expression System

A GAF domain of the cyanobacteriochrome (CBCR) Cha6605_4344 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). Cells appear to be off-yellow under normal light, and may be difficult to distinguish from a negative control.

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 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 740
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1260
Illegal AgeI site found at 1350
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K1916103 short</partinfo>
-The Cha6605_4344g2 protein (see BBa_K1916003) under control of a strong pBAD promoter.
+A GAF domain of the cyanobacteriochrome (CBCR) Cha6605_4344 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). Cells appear to be off-yellow under normal light, and may be difficult to distinguish from a negative control.
+==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:K1916103_cha_induced_vs_uninduced.jpeg|500px|thumb|left|Fig.1 Comparison of pellets from a negative control strain (left) and from cells spun down 4 hrs after being induced (right).]]
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