Difference between revisions of "Part:BBa K1385001:Design"
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===Use=== | ===Use=== | ||
To Use this promoter, you need to co-transform with the Phycocyanobilin Plasmid such as BBa_K1017726. You also need the CcaR/CcaS part such as BBa_K360051. And a Ptet promoter driving your desired gene. Then you have to grow cultures in either green light (535nm) or broad spectrum light to produce high levels of TetR. | To Use this promoter, you need to co-transform with the Phycocyanobilin Plasmid such as BBa_K1017726. You also need the CcaR/CcaS part such as BBa_K360051. And a Ptet promoter driving your desired gene. Then you have to grow cultures in either green light (535nm) or broad spectrum light to produce high levels of TetR. | ||
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| + | This is the plasmid we used it in: | ||
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| + | https://static.igem.org/mediawiki/2014/2/20/WashU_Hyb_map.png | ||
===Design Notes=== | ===Design Notes=== | ||
Revision as of 00:21, 14 October 2014
Hybrid CpcG2 promoter -> TetR
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 281
Illegal NheI site found at 304 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Use
To Use this promoter, you need to co-transform with the Phycocyanobilin Plasmid such as BBa_K1017726. You also need the CcaR/CcaS part such as BBa_K360051. And a Ptet promoter driving your desired gene. Then you have to grow cultures in either green light (535nm) or broad spectrum light to produce high levels of TetR.
This is the plasmid we used it in:
Design Notes
G-box, hybrid promoter ptrc1O The spacer region between TetR and cpcG2 can be modified in order to increase or decrease the RBS strength, as TetR start codon is a ways down from the end of the promoter. The region it is so far is because our primers when assembling the plasmid had troublesome secondary structures closer to the promoter and start codon, so we needed a spacer.
The reason we made it an inverter was because we wanted to turn off transcription in the light. We wanted transcription of our target gene to only be in the dark as our big picture is to use this in organisms that can both photosynthesize and fix nitrogen as separate processes. By separating the two into light/dark cycles, nitrogenase will be sheltered from high amounts of cellular oxygen that is a product of photosynthesis.
Source
cpcG2 promoter Genes from PJT122 (Tabor lab)
TetR from a plasmid Ptet-pp* available in Moon Lab, at Washington University in St. Louis
Trc1O from a plasmid pSL2264 available in Pakrasi Lab, at Washington University in St. Louis
References
Yuu Hirose et al (2008) "Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein." PNAS vol. 105: 9528–9533.
Tabor, J. J. et al.(2010), " Multichromatic Control of Gene Expression in Escherichia coli", J. Mol. Biol. , doi:10.1016/j.jmb.2010.10.038
Hirose, Narikawa, et al. "Cyanobacteriochrome CcaS regulates phycoerythrin accumulation in Nostoc punctiforme, a group II chromatic adapter" PNAS May 11, 2010 vol. 107 no. 19