Difference between revisions of "Part:BBa K1916100"
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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. | 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.]] | [[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.]] | ||
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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.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 131
Illegal SpeI site found at 686 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 125
Illegal SpeI site found at 686 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 65
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 131
Illegal SpeI site found at 686 - 25INCOMPATIBLE 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 - 1000COMPATIBLE WITH RFC[1000]