270 likes | 391 Views
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).
E N D
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
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-D-thiogalactopyranoside (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 • 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 aTchigh tetR High tetRlow 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 • 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