Creating a steady rate of state differentiation with single-strand

mispairing and fimE

Caltech 2008 iGEM ProjectAllen Lin

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

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

fimE details

fimE sensitivity

fimE binding site

Previous Experiment

Methods (1)

Cell line

Methods (2)

pBAD system: http://tools.invitrogen.com/content/sfs/brochures/710_01619_pBAD_bro.pdf

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), resulting

in pTSH14.

SSM

Methods

SSM switch frequency

Problem: Low, but not None

Solution: Add stop codon

Hin system