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

2. Projects Steps fimE SSM 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:

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), resulting in pTSH14.

11. SSM

13. Methods

14. SSM switch frequency

15. Problem: Low, but not None

16. Solution: Add stop codon

17. Hin system