Presentation is loading. Please wait.

Presentation is loading. Please wait.

Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng.

Similar presentations


Presentation on theme: "Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng."— Presentation transcript:

1 Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

2 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)

3 How Do Cells Repond? Transcription Network Activates gene in DNA Signal causes new proteins to be produced Slower than transduction

4 Shen-Orr et al. 2002

5 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

6 Gene Introduction Blunt Arrow—Repression Pointy Arrow—Activation E.G. If A high, then B low, C high, G low and steady state

7 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

8 Goal of Work Create customized gene networks to implement different logic circuits Input – Chemical concentration Output – Fluorescent protein (GFP)

9 Creating the Genes 3 prokaryotic transcription regulator proteins –LacI Modulated by isopropyl B-D- thiogalactopyranoside (IPTG) –TetR Modulated by anhydrotetracycline (aTc) –λ cI

10 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

11 Creating the Genes Promoters and protein coding regions were combined to create functional genes Sticky ends can be connected

12 Creating the Plasmid Plasmid – Circular DNA Each has 3 of the created genes Total of 125 different possible plasmids

13 Creating the Plasmid GFP gene included as an output signal -lite – tagged for degradation –Reduce toxicity and over expression

14 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”

15 Results Certain cells showed logical response E.G. NIF, NAND, NOR, AND

16 Results Same connectivity, different logic

17 Results Only up to 2.5% or 7% of the cells responded No set threshold

18 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

19 Second Results

20 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

21 Second Results Also possible—Change promoter and connectivity, but logic stays the same

22 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

23 Discussion Lac - red Line High aTc  high tetR High tetR  low λ cI Low λ cI  high GFP BUT low GFP observed

24 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

25 Discussion Elowitz and Leibler, 2000

26 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

27 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


Download ppt "Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng."

Similar presentations


Ads by Google