Transcript Combinatorial Synthesis of Genetic Networks

Combinatorial Synthesis of
Genetic Networks
Guet et. al.
Andrew Goodrich
Charles Feng
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)
How Do Cells Repond?
• Transcription Network
• Activates gene in DNA
• Signal causes new proteins to be
produced
• Slower than transduction
Shen-Orr et al. 2002
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
Gene Introduction
• Blunt Arrow—Repression
• Pointy Arrow—Activation
• E.G. If A high, then B low, C high, G low
and steady state
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
Goal of Work
• Create customized gene networks to
implement different logic circuits
• Input – Chemical concentration
• Output – Fluorescent protein (GFP)
Creating the Genes
• 3 prokaryotic transcription regulator
proteins
– LacI
• Modulated by isopropyl B-Dthiogalactopyranoside (IPTG)
– TetR
• Modulated by anhydrotetracycline (aTc)
– λ cI
Creating the Genes
• 5 Promoter regions
– 2 repressed by LacI (PL1 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
Creating the Genes
• Promoters and protein coding regions
were combined to create functional
genes
• Sticky ends can be connected
Creating the Plasmid
• Plasmid – Circular DNA
• Each has 3 of the created genes
• Total of 125 different possible plasmids
Creating the Plasmid
• GFP gene included as an output signal
• -lite – tagged for degradation
– Reduce toxicity and over expression
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”
Results
• Certain cells showed logical response
• E.G. NIF, NAND, NOR, AND
Results
• Same connectivity, different logic
Results
• Only up to 2.5% or 7% of the cells
responded
• No set threshold
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
Second Results
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
Second Results
• Also possible—Change promoter and
connectivity, but logic stays the same
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
Discussion
• Lac- red Line
High aTchigh tetR
High tetRlow λ cI
Low λ cI high GFP
BUT low GFP
observed
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
Discussion
Elowitz and Leibler, 2000
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
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