27
Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

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

Embed Size (px)

Citation preview

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

Combinatorial Synthesis of Genetic Networks

Guet et. al.

Andrew GoodrichCharles Feng

Page 2: 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)

Page 3: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

How Do Cells Repond?

• Transcription Network

• Activates gene in DNA

• Signal causes new proteins to be produced

• Slower than transduction

Page 4: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

Shen-Orr et al. 2002

Page 5: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

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

Page 6: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

Gene Introduction

• Blunt Arrow—Repression

• Pointy Arrow—Activation

• E.G. If A high, then B low, C high, G low and steady state

Page 7: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

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

Page 8: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

Goal of Work

• Create customized gene networks to implement different logic circuits

• Input – Chemical concentration

• Output – Fluorescent protein (GFP)

Page 9: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

Creating the Genes

• 3 prokaryotic transcription regulator proteins– LacI

• Modulated by isopropyl B-D-thiogalactopyranoside (IPTG)

– TetR• Modulated by anhydrotetracycline (aTc)

– λ cI

Page 10: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

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

Page 11: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

Creating the Genes

• Promoters and protein coding regions were combined to create functional genes

• Sticky ends can be connected

Page 12: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

Creating the Plasmid

• Plasmid – Circular DNA

• Each has 3 of the created genes

• Total of 125 different possible plasmids

Page 13: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

Creating the Plasmid

• GFP gene included as an output signal

• -lite – tagged for degradation– Reduce toxicity and over expression

Page 14: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

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”

Page 15: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

Results

• Certain cells showed logical response

• E.G. NIF, NAND, NOR, AND

Page 16: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

Results

• Same connectivity, different logic

Page 17: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

Results

• Only up to 2.5% or 7% of the cells responded

• No set threshold

Page 18: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

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

Page 19: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

Second Results

Page 20: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

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

Page 21: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

Second Results

• Also possible—Change promoter and connectivity, but logic stays the same

Page 22: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

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

Page 23: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

Discussion

• Lac- red LineHigh aTchigh tetR

High tetRlow λ cI

Low λ cI high GFP

BUT low GFP observed

Page 24: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

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

Page 25: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

Discussion

Elowitz and Leibler, 2000

Page 26: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

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

Page 27: Combinatorial Synthesis of Genetic Networks Guet et. al. Andrew Goodrich Charles Feng

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