Combinatorial Synthesis of Genetic Networks
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Combinatorial Synthesis of Genetic Networks
Guet et. al.
Andrew GoodrichCharles Feng
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How Do Cells Repond?
• Signal Transduction Network
• Proteins activate in a chain (phosphorylation)
• E.G. E. Coli swimming to aspartate
D. Bray, Proc. Natl. Acad. Sci. U.S.A. 99, 7 (2002)
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How Do Cells Repond?
• Transcription Network
• Activates gene in DNA
• Signal causes new proteins to be produced
• Slower than transduction
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Shen-Orr et al. 2002
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Gene Introduction
• Promoter—Controls production of protein
• Structural Gene—Controls which protein is produced
http://upload.wikimedia.org/wikipedia/commons/4/42/Lac_operon.png
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Gene Introduction
• Blunt Arrow—Repression
• Pointy Arrow—Activation
• E.G. If A high, then B low, C high, G low and steady state
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Combinatorial Synthesis
• Very similar to directed evolution
• Large number of different gene networks are created (called a library)
• Library is then screened for desired feature
• Process can then be iterated with new starting point
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Goal of Work
• Create customized gene networks to implement different logic circuits
• Input – Chemical concentration
• Output – Fluorescent protein (GFP)
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Creating the Genes
• 3 prokaryotic transcription regulator proteins– LacI
• Modulated by isopropyl B-D-thiogalactopyranoside (IPTG)
– TetR• Modulated by anhydrotetracycline (aTc)
– λ cI
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Creating the Genes
• 5 Promoter regions– 2 repressed by LacI (PL
1 and PL2)
– 1 repressed by TetR (PT)
– 1 repressed by λ cI (Pλ-)
– 1 activated by λ cI (Pλ+)
• Gives a total of 15 possible genes
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Creating the Genes
• Promoters and protein coding regions were combined to create functional genes
• Sticky ends can be connected
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Creating the Plasmid
• Plasmid – Circular DNA
• Each has 3 of the created genes
• Total of 125 different possible plasmids
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Creating the Plasmid
• GFP gene included as an output signal
• -lite – tagged for degradation– Reduce toxicity and over expression
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Experimental Procedure
• Plasmids transformed into E. Coli
• 2 strains of E.Coli, +/- wild type LacI
• Each clone grown under 4 conditions– +/- IPTG, +/- aTc (regulator proteins)
• GFP expression monitored over time
• Identify “logical circuits”
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Results
• Certain cells showed logical response
• E.G. NIF, NAND, NOR, AND
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Results
• Same connectivity, different logic
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Results
• Only up to 2.5% or 7% of the cells responded
• No set threshold
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Second Procedure
• 30 clones of different logical behaviors were retransformed and sequenced
• Following table is Lac- E.Coli host
• Different logical circuits possible
• Outputs not always full on or full off
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Second Results
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Second Results
• Replacing one of the promotors can change the logic
• E.G. Pλ+ to PT changes logic from ON to
NIF or NAND
• E.G. PL1 creates NOR
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Second Results
• Also possible—Change promoter and connectivity, but logic stays the same
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Discussion
• Can create many different logic circuits with these simple pieces
• Offers an evolutionary shortcut—change network instead of single gene
• Logic depends on both connectivity and promoters
• Output not always predictable
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Discussion
• Lac- red LineHigh aTchigh tetR
High tetRlow λ cI
Low λ cI high GFP
BUT low GFP observed
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Discussion
• Autoregulation difficult to predict
• In this diagram, lac represses itself
• Steady state enough to repress tet?
• Boolean on/off model neglects intracellular effects and changes
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Discussion
Elowitz and Leibler, 2000
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Future Possibilities
• Biological Computers– Very far off, but groundwork showing
• More complicated behaviors, including switches, sensors and oscillators
• Combinatorial techniques applied to proteins instead of gene networks
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References
1. Guet et. al. Science. 296, 1466 (2002)
2. D. Bray, Proc. Natl. Acad. Sci. U.S.A. 99, 7 (2002)
3. Shenn-Orr et. al. 2002
4. Elowitz and Leibler, 2000