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 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 4

Sense the environment Implement novel DNA degradation kit. 5

 Dark Works!  Lots of nutrition  Higher temperature  Uniform QS Fermentor BOOM!!!  Light  No nutrition  Lower temperature  Foreign QS 6 Environment

Cph1 Light receptor EnvZ P Temperature sensor Quorum Sensor Sensing AHL Molecule Module 7 High Sensitivity

RB cI S P ompC Lambda CI System c I bindi ng RB goi s ite S P L OFF fim gfp Inversion Ptrc* FimE System Processor ON gfp Module Ptrc* 8 Tight Regulation

Cell BOOM!!! lysis DNA degradation Lysis Module 9 High Performance

Processor Sensor Lysis Module Module Module 10

Processor Sensor Lysis Module Module Module 11

Light signal system Light signal system Temperature signal system Quorum sensing system 12

Chop coli prefers darkness over light 13

Signal transduction 14

50000 Light Relative Fluorescence 40000 30000 20000 Dark 10000 Control 0 0 5 10 Cultivation Time (Hour) Darkness represses the gene expression 15

Opaque Transparent 16

Relative Fluorescence 60000 Low Conc. Glucose 40000 20000 High Conc. Glucose Control 0 0 4 8 12 Cultivation Time (Hour) High concentration of sugar represses the gene expression. 17

Relative Fluorescence 80000 Low Conc. Arabinose 60000 40000 High Conc. Arabinose 20000 Control 0 0 4 8 12 Cultivation Time (Hour) High concentration of sugar represses the gene expression. 18

Light signal system Temperature signal system Temperature signal system Quorum sensing system 19

Riboswitch 37℃ 20

21 Olga, Cell (2011)

18000 10000 30 ⁰ C 9000 16000 Relative Fluorescence Relative Fluorescence 8000 14000 7000 12000 6000 10000 5000 8000 37 ⁰ C 4000 6000 3000 4000 2000 2000 1000 0 0 0 5 10 15 20 25 0 5 10 15 20 25 Cultivation Time (Hour) Cultivation Time (Hour) High temperature repress the gene expression. 22

Light signal system Temperature signal system Quorum sensing system Quorum sensing system 23

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Processor Sensor Lysis Module Module Module 25

Processor Sensor Lysis Module Module Module 26

To prevent the leaky expression To avoid undesired cell death during inoculum preparation 27

cI control system cI control system FimE inversion system 28

Darkness Cph1 EnvZ P P CI OmpR RBS cI RNAP P OmpC (+) OmpR cI binding GOI RBS site P L RNAP Transcription is blocked ! 29

Light Cph1 EnvZ P EnvZ RBS cI P OmpC (+) OmpR POI POI POI cI binding GOI RNAP RBS site P L Transcription begins ! 30

50000 Early- Log Relative Fluorescence Unit Phase 40000 1 Mid Log 3 30000 5 Phase 20000 Late Log Phase 10000 Control 0 0 4 8 Cultivation Time (Hour) CI control system can switch ON gene expression with 1h duration time 31

cI control system FimE inversion system FimE inversion system 32

OFF gfp IRR IRL P trc FimE P trc ON gfp IRR IRL 33

Processor Sensor Lysis Module Module Module 34

Processor Sensor Lysis Module Module Module 35

Lytic cassette RBS Dpn cassette Holin cassette Causes cell lysis Chops up genetic material 36

Cell Lysis Module Cell Lysis Module DNA Degradation Module 37

Holin Chop coli 38

Boom!! Holin Endolysin Boom!! Chop coli 39

RNAP RNAP RNAP RNAP Cell lysis, but DNA remnants 40

Cell Lysis Module DNA Degradation Module DNA Degradation Module 41

Dpn I Chop coli 42

Dpn I Chop coli DNA is chopped, but no cell lysis 43

Cph1 EnvZ P EnvZ RBS cI P OmpC OmpR CI binding Dpn Holin RBS RNAP Riboswitch site P L Holin High Temp Low Temp DNA Chopped ! No Cell Burst Cell Burst ! 44

Chop coli Light ON & Low temperature 45

Cell lysis & No DNA left 46

 Light Holin &  fim E  Temp. Dpn QS – search – need to  cI for universal Q optimize S on progress expression 47

Combine the temperature, light and quorum sensing signal with the lysis circuit 48

AHL Lytic Pcp cph1 fim E Cassette ho-pcyA Pompc cI phyB-PIF3 49

Combine the temperature, light and quorum sensing signal with the lysis circuit Industrially used microbes will have the lysis circuit 50

Essential for genetically modified microbes As much important as promoter or gene Reduces the fears around GMOs One best way to support Synthetic Biology 51

When synthetic microbes are to be used in environment , they should carry the programmed cell death system to avoid risks 52

Is Better Than Cure!! 53

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Special lecture High School Students Non Biologists Workshop

Media Attention We published an article in Korean language to introduce synthetic biology and iGEM competition to all universities in Korea.

Dr. Sung Kuk, Lee Dr. Cheol-Min, Ghim Dr. Yoon-Kyoung, Cho 57

Thank you 58