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Creating a steady rate of state differentiation with single-strand mispairing and fimE

Creating a steady rate of state differentiation with single-strand mispairing and fimE. Caltech 2008 iGEM Project Allen Lin. Projects Steps. fimE Replicate system in Kealing’s lab Add extra IRL left to native IRL, determine what happens SSM

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Creating a steady rate of state differentiation with single-strand mispairing and fimE

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  1. Creating a steady rate of state differentiation with single-strand mispairing and fimE Caltech 2008 iGEM Project Allen Lin

  2. Projects Steps • fimE • Replicate system in Kealing’s lab • Add extra IRL left to native IRL, determine what happens • SSM • Replicate system in Woude’s lab, using gfp instead of lacZ • Either: • Add fimE gene downstream of gfp, and remove PBAD • Add a gene that transcribes arabdinose , and use original vector

  3. What if fimE only uses closest IRL? • hin system in Salomella • Can we use this system in E. coli? • If so, then we can: • Have two SSM systems, one controlling transcription of fimE and a hin repressor, the other controlling transcription of hin system and a fimE repressor • Chances both are turned on are 10-6 • For our system, okay if both systems are turned on simultaneously ; just don’t want this to happen for a majority of cells

  4. fimE details

  5. fimE sensitivity

  6. fimE binding site

  7. Previous Experiment

  8. Methods (1) Cell line

  9. Methods (2) pBAD system: http://tools.invitrogen.com/content/sfs/brochures/710_01619_pBAD_bro.pdf

  10. The fim invertible region in its native phase ‘‘off’’ (IRL)orientation was PCR-amplified and cloned into the BamHI/EcoRI-digested pPROBE-NT (Miller et al., 2000), resultingin pTSH14.

  11. SSM

  12. Methods

  13. SSM switch frequency

  14. Problem: Low, but not None

  15. Solution: Add stop codon

  16. Hin system

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