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

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 (P L 1 and P L 2 ) –1 repressed by TetR (P T ) –1 repressed by λ cI (P λ - ) –1 activated by λ cI (P λ + ) 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. P λ + to P T changes logic from ON to NIF or NAND E.G. P L 1 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 Elowitz and Leibler, 2000