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ChoCoLate
[Chop – E. coli – Late]
Team UNIST_Korea
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Yu Yu-Li Lim, , Lee Ee Ee Seul, Shin in Jae Sung, g, Yoo Bo Bo Keun, , Song
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ChoCoLate [Chop E. coli Late] Team UNIST_Korea 1 Ee Ee Seul, - - PowerPoint PPT Presentation
ChoCoLate [Chop E. coli Late] Team UNIST_Korea 1 Ee Ee Seul, - - PowerPoint PPT Presentation
ChoCoLate [Chop E. coli Late] Team UNIST_Korea 1 Ee Ee Seul, Shin in Jae Sung, g, Yoo Bo Bo Keun, , Song Yu Yu-Li Lim, , Lee 2 Risk of accidental escape Risk of open test in environment Risk of deliberate misuse 3 Although
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Risk of open test in environment
Risk of accidental escape Risk of deliberate misuse
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Although there are many self-lysis devices, they didn’t concern DNA degradation.
UNIST_KOREA Team designed Novel programmed cell death device that chops up DNA
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Sense the environment
Implement novel DNA degradation kit.
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- Dark
- Lots of nutrition
- Higher temperature
- Uniform QS
- Light
- No nutrition
- Lower temperature
- Foreign QS
BOOM!!!
Works!
Fermentor Environment
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Sensor Module
High Sensitivity
Temperature sensor Light receptor Quorum Sensing Molecule
Cph1
EnvZ P
AHL
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Processor Module
Tight Regulation
PompC
RB S
cI
RB S c I bindi ng s ite
goi
PL
Lambda
CI
System
FimE gfp Ptrc* OFF gfp Ptrc* ON
fim
Inversion System
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High Performance
DNA degradation Cell lysis
BOOM!!!
Lysis Module
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Sensor Module Processor Module Lysis Module
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Sensor Module Processor Module Lysis Module
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Light signal system Light signal system Temperature signal system Quorum sensing system
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Chop coli prefers darkness over light
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Signal transduction
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Darkness represses the gene expression
10000 20000 30000 40000 50000 5 10
Cultivation Time (Hour) Relative Fluorescence
Light Dark Control
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Opaque Transparent
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High concentration of sugar represses the gene expression.
20000 40000 60000 4 8 12
Relative Fluorescence Cultivation Time (Hour) Low Conc. Glucose High Conc. Glucose Control
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High concentration of sugar represses the gene expression.
20000 40000 60000 80000 4 8 12
Low Conc. Arabinose High Conc. Arabinose Control Relative Fluorescence Cultivation Time (Hour)
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Light signal system Temperature signal system Quorum sensing system Temperature signal system
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Riboswitch
37℃
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Olga, Cell (2011)
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1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 5 10 15 20 25 2000 4000 6000 8000 10000 12000 14000 16000 18000 5 10 15 20 25
Relative Fluorescence Relative Fluorescence Cultivation Time (Hour) Cultivation Time (Hour)
High temperature repress the gene expression.
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30 ⁰ C 37 ⁰ C
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Light signal system Temperature signal system Quorum sensing system Quorum sensing system
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!
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Sensor Module Processor Module Lysis Module
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Sensor Module Processor Module Lysis Module
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To prevent the leaky expression To avoid undesired cell death during inoculum preparation
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FimE inversion system
cI control system cI control system
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Cph1 EnvZ P
OmpR
Darkness
POmpC (+)
RBS
cI
RBS
cI binding site
GOI
PL
Transcription is blocked !
OmpR
P
RNAP
CI
RNAP
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Cph1
RBS
cI binding site
GOI
PL POmpC (+)
RBS
cI EnvZ EnvZ P
OmpR
RNAP POI POI POI
Transcription begins !
Light
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Early- Log Phase Mid Log Phase Late Log Phase Control
Relative Fluorescence Unit
CI control system can switch ON gene expression with 1h duration time
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10000 20000 30000 40000 50000 4 8 1 3 5
Cultivation Time (Hour)
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FimE inversion system
cI control system
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FimE inversion system
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gfp
IRR IRL
Ptrc
OFF
gfp
IRR IRL
Ptrc
ON
FimE
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Sensor Module Processor Module Lysis Module
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Sensor Module Processor Module Lysis Module
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RBS
Lytic cassette
Holin cassette Dpn cassette
Causes cell lysis
Chops up genetic material
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Cell Lysis Module
DNA Degradation Module
Cell Lysis Module
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Chop coli
Holin
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Chop coli
Holin
Endolysin
Boom!! Boom!!
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RNAP RNAP RNAP RNAP
Cell lysis, but DNA remnants
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Cell Lysis Module
DNA Degradation Module DNA Degradation Module
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Chop coli
DpnI
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Chop coli
DpnI
DNA is chopped, but no cell lysis 43
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CI binding site
Dpn Holin
PL
Riboswitch RBS
Cph1
EnvZ P
EnvZ
OmpR
POmpC
RBS
cI
RNAP
High Temp DNA Chopped ! No Cell Burst
Holin
Low Temp
Cell Burst !
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Chop coli
Light ON & Low temperature
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Cell lysis & No DNA left
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Light Temp. QS – search
for universal Q S on progress
fimE cI Holin & Dpn – need to
- ptimize
expression
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Combine the temperature, light and quorum sensing signal with the lysis circuit
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Pcp ho-pcyA cph1 Pompc cI
fimE
phyB-PIF3
AHL Lytic Cassette
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Combine the temperature, light and quorum sensing signal with the lysis circuit Industrially used microbes will have the lysis circuit
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Essential for genetically modified microbes As much important as promoter or gene Reduces the fears around GMOs One best way to support Synthetic Biology
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When synthetic microbes are to be used in environment, they should carry the programmed cell death system to avoid risks
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Is Better Than
Cure!!
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Special lecture High School Students Workshop Non Biologists
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Media Attention
We published an article in Korean language to introduce synthetic biology and iGEM competition to all universities in Korea.
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- Dr. Sung Kuk, Lee
- Dr. Yoon-Kyoung, Cho
- Dr. Cheol-Min, Ghim
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Thank you
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