Difference between revisions of "Part:BBa K1916100"

Revision as of 06:53, 25 October 2016

Ara-Inducible NpF2164g5 Expression System

A GAF domain of the cyanobacteriochrome (CBCR) NpF2164 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). NpF2164g5 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.

Pellets of cells expressing NpF, spun down 4 hrs after being induced by different concentrations of arabinose.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 131
Illegal SpeI site found at 686
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 125
Illegal SpeI site found at 686
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 686
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 131
Illegal SpeI site found at 686
Illegal NgoMIV site found at 1182
Illegal AgeI site found at 1272
1000
COMPATIBLE WITH RFC[1000]

@@ Line 6: / Line 6: @@
 ==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.
 [[Image:K1916100_npf_induction.jpeg|600px|thumb|left|Pellets of cells expressing NpF, spun down 4 hrs after being induced by different concentrations of arabinose.]]