SLIDE 1
Does God play dice with the cell? Does God play dice with the cell? Does God play dice with the cell? Does God play dice with the cell? Does God play dice with the cell? Does God play dice with the cell? Does God play dice with the cell? Does God play dice with the cell?
HKUSTers HKUSTers HKUSTers HKUSTers HKUSTers HKUSTers HKUSTers HKUSTers
Hong Kong University of Science & Technology Hong Kong University of Science & Technology iGEM iGEM 2008 2008
SLIDE 2 Integration of Integration of Integration of Integration of … … … …
Students from Students from Students from Students from Students from Students from Students from Students from
Biochemistry Biochemistry Biochemistry Biochemistry Biochemistry Biochemistry Biochemistry
Chemistry Chemistry Chemistry Chemistry Chemistry Chemistry Chemistry
Physics Physics Physics Physics Physics Physics Physics
Computer Science Computer Science Computer Science Computer Science Computer Science Computer Science Computer Science
Computer Computer Computer Computer Computer Computer Computer Engineering Engineering Engineering Engineering Engineering Engineering Engineering Engineering & & & & & & & &
High School High School High School High School High School High School High School Science class Science class Science class Science class Science class Science class Science class Science class Instructor: Instructor: Instructor: Instructor: Instructor: Instructor: Instructor: Instructor:
- Dr. King L. Chow
- Dr. King L. Chow
- Dr. King L. Chow
- Dr. King L. Chow
- Dr. King L. Chow
- Dr. King L. Chow
- Dr. King L. Chow
- Dr. King L. Chow
SLIDE 3
SLIDE 4 Randomizer Randomizer Randomizer Randomizer Randomizer Randomizer Randomizer Randomizer
Randomness – – Randomizer Randomizer
Generate 0 or 1 randomly
SLIDE 5 Application Application Application Application Application Application Application Application
Statistical Statistical Statistical Statistical Statistical Statistical Statistical sampling & analysis: sampling & analysis: inputs inputs
Simulation Simulation Simulation Simulation Simulation Simulation Simulation of real and complex process,
- f real and complex process,
such as biological process: such as biological process: noise noise
SLIDE 6 Outline Outline Outline Outline Outline Outline Outline Outline
Design Design Design Design Design Design Design & c & c & c & c & c & c & c & construction
- nstruction
- nstruction
- nstruction
- nstruction
- nstruction
- nstruction
- nstruction
- Simulation
Simulation Simulation Simulation Simulation Simulation Simulation Simulation
Lab work Lab work Lab work Lab work Lab work Lab work Lab work
Further extension Further extension Further extension Further extension Further extension Further extension Further extension
SLIDE 7
A Switch A Switch A Switch A Switch A Switch A Switch A Switch A Switch
[1] [0]
either right [0], either right [0], either right [0], either right [0], either right [0], either right [0], either right [0], either right [0], ……… ……… ……… ……… ……… ……… ……… ……… … … … … … … … ….with randomness .with randomness .with randomness .with randomness .with randomness .with randomness .with randomness .with randomness
SLIDE 8
A Switch A Switch A Switch A Switch A Switch A Switch A Switch A Switch
[1] [0]
either right [0], either right [0], either right [0], either right [0], either right [0], either right [0], either right [0], either right [0], ……… ……… ……… ……… ……… ……… ……… ……… … … … … … … … ….with randomness .with randomness .with randomness .with randomness .with randomness .with randomness .with randomness .with randomness
SLIDE 9
A Switch A Switch A Switch A Switch A Switch A Switch A Switch A Switch
[1] [0]
… … … … … … … …..or left [1]. ..or left [1]. ..or left [1]. ..or left [1]. ..or left [1]. ..or left [1]. ..or left [1]. ..or left [1]. … … … … … … … ….with randomness .with randomness .with randomness .with randomness .with randomness .with randomness .with randomness .with randomness
SLIDE 10
A Switch A Switch A Switch A Switch A Switch A Switch A Switch A Switch
[1] [0]
… … … … … … … …..or left [1]. ..or left [1]. ..or left [1]. ..or left [1]. ..or left [1]. ..or left [1]. ..or left [1]. ..or left [1]. … … … … … … … ….with randomness .with randomness .with randomness .with randomness .with randomness .with randomness .with randomness .with randomness
SLIDE 11
A Switch A Switch A Switch A Switch A Switch A Switch A Switch A Switch
[1] [0]
… … … … … … … …..or left [1]. ..or left [1]. ..or left [1]. ..or left [1]. ..or left [1]. ..or left [1]. ..or left [1]. ..or left [1]. … … … … … … … ….with randomness .with randomness .with randomness .with randomness .with randomness .with randomness .with randomness .with randomness
SLIDE 12 T7 polymerase Promoter Terminator Ribosome binding site TetR repressor Green/Red fluorescence protein
Genetic circuit Genetic circuit Genetic circuit Genetic circuit
START START START START
SLIDE 13 T7 polymerase Promoter Terminator Ribosome binding site TetR repressor Green/Red fluorescence protein
Genetic circuit Genetic circuit Genetic circuit Genetic circuit
START START START START
SLIDE 14 T7 polymerase Promoter Terminator Ribosome binding site TetR repressor Green/Red fluorescence protein
Genetic circuit Genetic circuit Genetic circuit Genetic circuit
START START START START
SLIDE 15 T7 polymerase Promoter Terminator Ribosome binding site TetR repressor Green/Red fluorescence protein
Genetic circuit Genetic circuit Genetic circuit Genetic circuit
START START START START
SLIDE 16 T7 polymerase Promoter Terminator Ribosome binding site TetR repressor Green/Red fluorescence protein
Genetic circuit Genetic circuit Genetic circuit Genetic circuit
START START START START Transcription blocked Transcription blocked Transcription blocked Transcription blocked
SLIDE 17 Genetic circuit Genetic circuit Genetic circuit Genetic circuit
START START START START Transcription blocked Transcription blocked Transcription blocked Transcription blocked
T7 polymerase Promoter Terminator Ribosome binding site cI434 repressor Green/Red fluorescence protein
SLIDE 18 When you have too many coins, a single bacterium will still show a When you have too many coins, a single bacterium will still show a When you have too many coins, a single bacterium will still show a When you have too many coins, a single bacterium will still show a When you have too many coins, a single bacterium will still show a When you have too many coins, a single bacterium will still show a When you have too many coins, a single bacterium will still show a When you have too many coins, a single bacterium will still show a mix of colors. mix of colors. mix of colors. mix of colors. mix of colors. mix of colors. mix of colors. mix of colors. But h But h But h But h But h But h But h But how many targets
- w many targets
- w many targets
- w many targets
- w many targets
- w many targets
- w many targets
- w many targets can a polymerase act on
can a polymerase act on can a polymerase act on can a polymerase act on can a polymerase act on can a polymerase act on can a polymerase act on can a polymerase act on? ? ? ? ? ? ? ? If there are more than one, 1 and 0 are both read If there are more than one, 1 and 0 are both read If there are more than one, 1 and 0 are both read If there are more than one, 1 and 0 are both read If there are more than one, 1 and 0 are both read If there are more than one, 1 and 0 are both read If there are more than one, 1 and 0 are both read If there are more than one, 1 and 0 are both read... ... ... ... ... ... ... ...
Integration Integration Integration Integration
SLIDE 19
Integration system Integration system Integration system Integration system
SLIDE 20 Lambda InCh a simple E. coli plasmid- chromosome shuttle system Boyd, D., Weiss, D. S., Chen, J. C. & Beckwith, J
Integration system Integration system Integration system Integration system
SLIDE 21 Lysogen Lysogen Lysogen Lysogen Lysogen Lysogen Lysogen Lysogen will be killed at 42 will be killed at 42 will be killed at 42 will be killed at 42 will be killed at 42 will be killed at 42 will be killed at 42 will be killed at 42º º º º º º º ºC C C C C C C C
Lambda InCh a simple E. coli plasmid-chromosome shuttle system Boyd, D., Weiss, D. S., Chen, J. C. & Beckwith, J
Loop out Loop out Loop out Loop out Loop out Loop out Loop out Loop out
Integration system Integration system Integration system Integration system
SLIDE 22
Cell number Cell number Cell number Cell number
SLIDE 23 Predicted output Predicted output Predicted output Predicted output
START Transcription blocked
Interval (t1) Interval (t1) Delay (t2) Delay (t2)
T7 polymerase Promoter Terminator Ribosome binding site TetR repressor Green/Red fluorescence protein
SLIDE 24 Core Parameter Core Parameter Core Parameter Core Parameter
Interval t1:
– – between 2 T7-Polymerase Binding events; between 2 T7-Polymerase Binding events;
Delay t2:
– – until the repressor protein binds to the DNA until the repressor protein binds to the DNA and inhibits the opposite and inhibits the opposite direction direction; ;
==> ==> ratio t1/t2: the larger, the better.
ratio t1/t2: the larger, the better.
SLIDE 25
Parameters Plotted Parameters Plotted Parameters Plotted Parameters Plotted
For the total pool of cells, Define SuccessRate = # of distinct cells total # of cells Define Bias = # of (green - red) cells # of distinct cells
SLIDE 26 Overlapping promoter test Overlapping promoter test Overlapping promoter test Overlapping promoter test
5’ CCC CTATAGTGAGTCGTATTAATACGACTCACTATAG GGG 3’ GGG GATATCACTCAGCATAATTATGCTGAGTGATATC CCC
Overlapping promoter test 2 overlapped basepairs
Control Test 5’ TAATACGACTCACTATA 3’ ATTATGCTGAGTGATAT
Preliminary Test Preliminary Test Preliminary Test Preliminary Test
Truncated promoter test 5’ TAATACGACTCACTATA 3’ ATTATGCTGAGTGATAT
SLIDE 27 Overlapping promoter test Overlapping promoter test Overlapping promoter test Overlapping promoter test
5’ CCC CTATAGTGAGTCGTACTACGACTCACTATAG GGG 3’ GGG GATATCACTCAGCATGATGCTGAGTGATATC CCC
More overlapping promoter test 5 overlapped basepairs
5’ CCC CTATAGAGAGTCGTATTAATACGACTCTCTATAG GGG 3’ GGG GATATCTCTCAGCATAATTATGCTGAGAGATATC CCC
Weakened overlapped promoter (activity 0.13/1.00 Mutant/WT)
T7 promoter contacts essential for promoter activity in vivo Richard A.lkeda, Cathleen M.Ligman and Sakuntala Warshamana School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332-0400, USA
SLIDE 28 AraC/pBad AraC/pBad AraC/pBad AraC/pBad Induction Test Induction Test Induction Test Induction Test
Tight Regulation, Modulation, and High-Level Expression by Tight Regulation, Modulation, and High-Level Expression by Tight Regulation, Modulation, and High-Level Expression by Tight Regulation, Modulation, and High-Level Expression by Vectors Containing the Vectors Containing the Vectors Containing the Vectors Containing the Arabinose Arabinose Arabinose Arabinose PBAD Promoter PBAD Promoter PBAD Promoter PBAD Promoter LUZ-MARIA GUZMAN,1 DOMINIQUE BELIN,2 MICHAEL J. CARSON,3 AND JON BECKWITH1 *
SLIDE 29
Repressor Test Repressor Test Repressor Test Repressor Test
SLIDE 30 A A A B B B B B B B B B B B B
Further extension Further extension Further extension Further extension
Start Start Start Start Start Start Start Start Memorizer Memorizer
B B B B B B B B A A A A A A A A
Same outcome Same outcome Same outcome Same outcome Same outcome Same outcome Same outcome Same outcome
No response No response No response No response No response No response No response
Jackpot signal! Jackpot signal! Jackpot signal! Jackpot signal! Jackpot signal! Jackpot signal! Jackpot signal! Jackpot signal! Add Memorizer + Reporter Add Memorizer + Reporter Add Memorizer + Reporter Add Memorizer + Reporter Add Memorizer + Reporter Add Memorizer + Reporter Add Memorizer + Reporter Add Memorizer + Reporter
Randomizer Randomizer
SLIDE 31
Further extension Further extension Further extension Further extension
If it works successfully, If it works successfully, If it works successfully, If it works successfully, If it works successfully, If it works successfully, If it works successfully, If it works successfully, we can generate random binary numbers we can generate random binary numbers we can generate random binary numbers we can generate random binary numbers we can generate random binary numbers we can generate random binary numbers we can generate random binary numbers we can generate random binary numbers within a single bacterial cell. within a single bacterial cell. within a single bacterial cell. within a single bacterial cell. within a single bacterial cell. within a single bacterial cell. within a single bacterial cell. within a single bacterial cell. 10001110010000111110111011111101 10001110010000111110111011111101
SLIDE 32
Acknowledgement Acknowledgement Acknowledgement Acknowledgement
SLIDE 33
Q Q Q Q Q Q Q Q & A & A & A & A & A & A & A & A
