Ham et al inversion switch

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Ham et al inversion switch
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Inversion switch
Heritable inversion swith State determined by
sequence of inputs across generations
(time) Reporter records path traverse through
states
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Recombinases
Recognition sites 30 bp inverted repeating
sequences Distance between sites Several hundred
to 5kb
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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
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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.
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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.
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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

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

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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).

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