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Directed Evolution of a Genetic Circuit

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Directed Evolution of a Genetic Circuit. Balaji ... Ligate (BsaI sites) Pfu PCR. Taq PCR. Ligase. Screen Mutants. Want: IPTG 0, EYFP 1. IPTG 1, EYFP 0 ... – PowerPoint PPT presentation

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Title: Directed Evolution of a Genetic Circuit


1
Directed Evolution of a Genetic Circuit
  • Balaji Srinivasan

2
Problem
  • Want to design genetic circuits
  • Understand naturally occuring networks
  • Bioengineering applications
  • But...
  • circuits that work on paper never work in the
    lab...
  • at least, not without optimization.

3
Making it work
  • Several options
  • Rational debugging (Weiss)
  • Combinatorial synthesis (Guet)
  • Directed evolution (this paper)

4
Goal Build Inverter
  • Simplest Logic Gate

B
5
Inverter Implementation
  • Two plasmid system
  • 2 regulatory proteins
  • IPTG input
  • EYFP reporter

6
Relevant Genetics
7
Nonfunctional at first
  • Leaky Plac ? product even w/o input
  • low cI still represses EYFP expression
  • Gate mismatch
  • Want IPTG 0 ? EYFP 1
  • Instead IPTG 0 interpreted as IPTG 1 from EYFP
    persp.
  • Very familiar to EEs (impedance matching)

Low IPTG input
But still no fluorescence
8
Solution 1 Rational Design
  • Reason that cI protein is too prevalent
  • Damage RBS ? reduce cI protein levels
  • Mutagenize Plac ? reduce leakiness
  • Mutagenize PR ? reduce cI effectiveness
  • This can be done (see Weiss)but its laborious

9
Solution 2 Evolve cI protein
  • Cut plasmid
  • PCR both pieces
  • Mutagenic (Taq) PCR for cI gene and RBS
  • Proofreading (Pfu) PCR for rest of plasmid
  • Ligate (BsaI sites)

Pfu PCR
Taq PCR
Ligase
10
Screen Mutants
  • Want
  • IPTG 0, EYFP 1
  • IPTG 1, EYFP 0

Jackpot!
IPTG low
IPTG high
11
Systems Analysis of Mutants
  • Can now plot inverter device curves
  • Compare to idealized inverter

12
Characterize Mutants
  • Sequence successful cI mutant genes
  • How did they solve the problem?
  • What does this mean structurally?

13
Structural Mutants
  • Many solutions
  • Stop codons in cI ? remove C-term dimerization
    domain entirely!
  • Disrupt start, RBS (reduce translation
    efficiency)
  • Synonymous mutations (change transc/transl?)
  • Protein-DNA interaction altered
  • N and C term dimerization altered
  • Model dimerization was another thing to tweak

14
Conclusion
  • Directed evolution potentially potent tool for
    tuning circuits that should work
  • Obvious research topics
  • Selections rather than screens (must be careful)
  • Multiplexing eliminate REAL cloning

15
References
  • Guet comb. synthesis
  • Elowitz repressilator
  • Collins bistable inverter
  • Hasty theory
  • Leibler everything
  • Barkai everything

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