Inverter

Part:BBa_K1385001:Design

Designed by: Benjamin Huang   Group: iGEM14_WashU_StLouis   (2014-10-07)
Revision as of 00:21, 14 October 2014 by Benjaminhuang (Talk | contribs) (→‎Use)

Hybrid CpcG2 promoter -> TetR


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 281
    Illegal NheI site found at 304
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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:

WashU_Hyb_map.png

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