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Ham et al inversion switch

Ham et al inversion switch. Inversion switch. Heritable inversion swith State determined by sequence of inputs across generations (time) Reporter records path traverse through states. Recombinases. Recognition sites 30 bp inverted repeating sequences Distance between sites

candace-coffey
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Ham et al inversion switch

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  1. Ham et al inversion switch

  2. Inversion switch Heritable inversion swith State determined by sequence of inputs across generations (time) Reporter records path traverse through states

  3. Recombinases Recognition sites 30 bp inverted repeating sequences Distance between sites Several hundred to 5kb

  4. Assumptions Single target Single copy of DNA target Irreversible flipping A single inversion of target sequence No cross talk Between invertases Sequential inversion N! possible ways of ordering N invertases in a sequence

  5. Optimization Independently induced Independent expression systems Minimize leakiness Tightly controlled expression systems Optimize translational efficiency – for expression Try several RBSs Sensitive reporting PCR, highly sensitive Therefore, bias against leakiness.

  6. Function Functions like an AND gate, with output contingent upon the sequence of inputs it had observed, the history of inputs. Number of possible invertase site configurations increases better than exponentially with the number of invertases. Every possible history of inputs is recordable in the state of the DNA output.

  7. Reality Reversible Single copy of DNA target Mirrored pairing Introduced hairpin structure that is hard to construct and sequence FimB – DNA interface There seems to be interference between recombinases Proof of concept States shown, but not in frequency expected; poor Fim transition

  8. Goal Tightly regulated, inducible, gene expression systems for molecular biology, since controlling gene expression in cells is essential for pathway investigation and manipulation

  9. Inducible promoters Proven [1,2] Leaky [3] RBS engineering Mitigate leakiness [4] Reduced expression [5] • IPTG-induced trc promoter (Ptrc) (Amann et al., 1988) • Arabinose-induced araBAD promoter (PBAD) systems (Guzman et al., 1995) • Non-induced basal expression can be significant • 4. Leaky expression can be mitigated by altering RBS (Guzman et al., 1995) • This often results in a reduced induced expression level as well

  10. Phage Int Inversion [1] Not leaky [2] Tight control [3] High induction level [4] Special context [5] FimE Unidirectional Inversion [6-9] Not leaky [de0coupled] Tight control High induction level No hosts or complex induction methods A Tightly Regulated Inducible Expression System Utilizing the fim Inversion Recombination Switch Timothy S. Ham,1 Sung Kuk Lee,2 Jay D. Keasling,1,2,3 Adam P. Arkin1,3 • Podhajska et al. (1985) • Decoupling the induction mechanism from the expression promoter • Tight control in the un-induced state • High induction level when induced (Sektas et al., 2001). • However, the Int/att system requires not only a specialized host containing an inducible int, but also a heat-shock-based induction method, which could make this system undesirable for certain applications. • Inversion of a 314-base pair (bp) DNA segment containing promoter • Two invertases, FimB and FimE (Klemm, 1986) • FimB is able to invert the DNA segment in both directions • FimE inverts from ‘‘on’’ to ‘‘off’’ (Blomfield et al., 1991; review in Blomfield, 2001).

  11. Tightly Regulated Inducible Expression System Utilizing the fim Inversion Switch Timothy S. Ham,1 Sung Kuk Lee,2 Jay D. Keasling,1,2,3 Adam P. Arkin1,3 Performs inversion from the PFR to PFL orientation PFL orientation: off (native), on (Arkin et al)

  12. Native ribosome binding site (RBS) for fimE was susceptible to sporadic, uninduced FimE expression by the leaky PBAD

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