Ham et al inversion switch

Heritable inversion swithState determined by sequence of inputs across generations (time)Reporter records path traverse through states

Inversion switch

Recognition sites30 bp inverted repeating sequences

Distance between sitesSeveral hundred to 5kb

Recombinases

Single targetSingle copy of DNA target

Irreversible flippingA single inversion of target sequence

No cross talkBetween invertases

Sequential inversionN! possible ways of ordering N invertases in a sequence

Assumptions

Independently inducedIndependent expression systems

Minimize leakinessTightly controlled expression systems

Optimize translational efficiency – for expressionTry several RBSs

Sensitive reportingPCR, highly sensitive

Therefore, bias against leakiness.

Optimization

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.

Function

ReversibleSingle copy of DNA target

Mirrored pairingIntroduced hairpin structure that is hard to construct and sequence

FimB – DNA interfaceThere seems to be interference between recombinases

Proof of conceptStates shown, but not in frequency expected; poor Fim transition

Reality

GoalTightly regulated, inducible, gene expression systems for molecular biology,

since controlling gene expression in cells is essential for pathway investigation and manipulation

1.IPTG-induced trc promoter (Ptrc) (Amann et al., 1988) 2.Arabinose-induced araBAD promoter (PBAD) systems (Guzman et al., 1995) 3.Non-induced basal expression can be significant4. Leaky expression can be mitigated by altering RBS (Guzman et al., 1995)5.This often results in a reduced induced expression level as well

Inducible promotersProven [1,2]

Leaky [3]

RBS engineeringMitigate leakiness [4]

Reduced expression [5]

1.Podhajska et al. (1985) 2.Decoupling the induction mechanism from the expression promoter3.Tight control in the un-induced state 4.High induction level when induced (Sektas et al., 2001).5.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.6.Inversion of a 314-base pair (bp) DNA segment containing promoter 7.Two invertases, FimB and FimE (Klemm, 1986)8.FimB is able to invert the DNA segment in both directions9.FimE inverts from ‘‘on’’ to ‘‘off’’ (Blomfield et al., 1991; review in Blomfield, 2001).

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 levelNo hosts or complex induction methodsA 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

PFL orientation: off (native), on (Arkin et al)

Performs inversion from the

PFR to PFLorientation

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

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