Reguli KAIST iGEM 2012 Regulation of E.coli Regulation of + - - PowerPoint PPT Presentation

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Reguli KAIST iGEM 2012 Regulation of E.coli Regulation of + - - PowerPoint PPT Presentation

May 19, 2023 370 likes •1.07k views

Reguli KAIST iGEM 2012 Regulation of E.coli Regulation of + E.coli Regu Regu Regulation + E.coli li li Prototype Mycobacterium Phage Bxb1, gp35(DNA integrase) & gp47(DNA excisionase) Target att B att P Mycobacterium Phage

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Reguli

KAIST iGEM 2012

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Regulation E.coli

  • f
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Regulation E.coli

  • f

+

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Regu li Regu + li Regulation E.coli

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Prototype

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Target att B att P att L att R att B att P Target Target

Mycobacterium Phage Bxb1, gp35(DNA integrase) & gp47(DNA excisionase)

Mycobacterium Phage Bxb1 Integrase/Excisionase complex Mycobacterium Phage Bxb1 Integrase

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att B att P Promoter GFP RFP att L att R GFP RFP

RFP GF P

Prototype : Design

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Prototype : Experimental Data - Cell Culture

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Prototype : Experimental Data - Confocal Microscopy

GFP initial + Integrase

uninduced induced

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140000 12hr 6hr 12hr 90000 6hr

RFP control GFP control

GFP initial

Prototype : Experimental Data - Fluorescent Intensity

MG1655 negative control GFP initial + Integrase GFP initial + Integrase : Induced

GFP GFP(RFU) (RFU) RFP(RFU) RFP(RFU)

Induction Induction

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RFP initial

12hr 40000 16000 12hr

GFP GFP(RFU) (RFU) RFP(RFU) RFP(RFU)

6hr 6hr

Induction Induction

Prototype : Experimental Data - Fluorescent Intensity

RFP control GFP control MG1655 negative control RFP initial + Integrase RFP initial + Integrase : Induced

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Reguli Module

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Reguli Module : Design Reguli Module

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Bacterial Flavin-containing Monooxygenase

Applicability Visibility

N-acyl homoserine lactone

Regulatory factor Reguli Module : Design

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Reguli Module : Mechanism

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pLuxR Promoter BBa_J23119 LuxR Bxb1 gp35 (Int) pSB4A5 Terminator BBa_B0015

pAutoIntegrase 5876bp

Reguli Module : Mechanism

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pLuxR Promoter BBa_J23119 LuxR Bxb1 gp35 (Int) pSB4A5 Terminator BBa_B0015 LuxR

pAutoIntegrase 5876bp

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pAuto 5832bp

LuxI pSB1C3 bFMO aiiA Bxb1 gp47 (Xis) attB attP Promoter BBa_J23119

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LuxI

pAuto 5832bp

pSB1C3 bFMO aiiA Bxb1 gp47 (Xis) LuxI Promoter BBa_J23119 attB attP

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LuxI AHL

pAuto 5832bp

pSB1C3 bFMO aiiA Bxb1 gp47 (Xis) LuxI Promoter BBa_J23119 attB attP

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LuxI pAutoIntegrase 5876bp pAuto 5832bp LuxR LuxR AHL

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LuxI LuxR/AHL complex LuxR LuxR AHL pAutoIntegrase 5876bp pAuto 5832bp

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pLuxR Promoter BBa_J23119 LuxR Bxb1 gp35 (Int) pSB4A5 Terminator BBa_B0015

pAutoIntegrase 5876bp

LuxR/AHL complex

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pLuxR Promoter BBa_J23119 LuxR Bxb1 gp35 (Int) pSB4A5 Terminator BBa_B0015

pAutoIntegrase 5876bp

LuxR/AHL complex

Bxb1 gp35

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Bxb1 gp35

LuxI pSB1C3 bFMO aiiA Bxb1 gp47 (Xis) attB

pAuto 5832bp

attP

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Bxb1 gp35

pAuto 5832bp

LuxI pSB1C3 bFMO aiiA Bxb1 gp47 (Xis) attB attP attL attR

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Indole Isatin

“BIO-INDIGO”

pAuto 5832bp

attL attR Inverted Promoter bFMO bFMO

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pAuto 5832bp

bFMO aiiA attL attR

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pAuto 5832bp

bFMO aiiA

Lactonase

attL attR

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pAuto 5832bp

bFMO aiiA attL attR

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pLuxR

pAutoIntegrase 5876bp

pAuto 5832bp

bFMO aiiA

Bxb1 gp47 (Xis)

attL attR

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pLuxR

pAutoIntegrase 5876bp

Bxb1 gp47

pAuto 5832bp

bFMO aiiA

Bxb1 gp47 (Xis)

attL attR

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Bxb1 gp47

pAuto 5832bp

bFMO aiiA

Bxb1 gp47 (Xis) Bxb1 gp35

attL attR

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pAuto 5832bp

bFMO aiiA

Bxb1 gp47 (Xis) Bxb1 gp35/47 complex Bxb1 gp35

attL attR

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pAuto 5832bp

attB attP bFMO aiiA

Bxb1 gp47 (Xis) Bxb1 gp35/47 complex Bxb1 gp35

attL attR

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pAuto 5832bp

LuxI pSB1C3 bFMO aiiA Bxb1 gp47 (Xis) attB attP Promoter BBa_J23119 pLuxR Promoter BBa_J23119 LuxR Bxb1 gp35 (Int) pSB4A5 Terminator BBa_B0015

pAutoIntegrase 5876bp

Reguli Module : Vector Design

low copy number high copy number

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Conventional Methods

endogenous synthetic

Promoter Binding site CDS Inducer

IPTG

ATP NADH

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Autonomous Regulation No Inducer

IPTG

Reguli Module : Advantages

ON OFF

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Reguli Module : Modeling

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Reguli Module : Modeling

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Reguli Module : Modeling - Prototype

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Reguli Module : Modeling - Prototype

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Reguli Module : Modeling - Prototype

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Reguli Module : Modeling - Prototype

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Reguli Module : Modeling - Prototype

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Reguli Module : Modeling - Reguli Module

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Reguli Module : Modeling - Reguli Module

Reguli Conventional

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Reguli Module : Modeling - Reguli Module

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Reguli Module : Experimental Data

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Reguli Module : Experimental Data

500bp 1kb 2kb 3kb pAutoSimple pAuto

pAutoIntegrase 5876bp

pAuto 5832bp

pAutoSimple 4217bp

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Reguli Module : Experimental Data

Inverted

Color change of host cells

0hr 3hr 6hr 9hr 15hr

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Reguli Module : Applications

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att B att P Promoter

Reguli Module - Highly applicable

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Bacteri-Guard

Environmental Applications

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Bacteri-Guard

Environmental Applications

pBacteri-Guard

Pollutant sensor vector Constitutive Promoter Scaveng er GFP Bxb1 gp47 (Xis) attB attP E.coli Genom e Sensor- induced promoter Bxb1 gp35 (Int)

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Bacteri-Guard

Environmental Applications

pBacteri-Guard

Pollutant sensor vector Constitutive Promoter Scaveng er GFP Bxb1 gp47 (Xis) attB attP E.coli Genom e Sensor- induced promoter Bxb1 gp35 (Int)

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Bacteri-Guard

Environmental Applications

pBacteri-Guard

Pollutant sensor vector Constitutive Promoter Scaveng er GFP Bxb1 gp47 (Xis) attB attP E.coli Genom e Sensor- induced promoter Bxb1 gp35 (Int)

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Bacteri-Guard

Environmental Applications

pBacteri-Guard

Pollutant sensor vector GFP Bxb1 gp47 (Xis) Scaveng er

pBacteri-Guard

Pollutant sensor vector Scaveng er GFP Bxb1 gp47 (Xis)

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Bacteri-Guard

Environmental Applications

pBacteri-Guard

Pollutant sensor vector Scaveng er GFP Bxb1 gp47 (Xis)

pBacteri-Guard

Pollutant sensor vector Scaveng er GFP Bxb1 gp47 (Xis)

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pollutants pollutants

Previous idea Bacteri-Guard

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Conclusion

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In this project, we iGEM 2012 KAIST,

Novel Biobrick parts Highly Manageable Biological System Environmental Applications

Conclusion Highly Manageable Biological System

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JUL 24th~27th, Talk with middle school students AUG 20th, Talk with high school students AUG 6th~10th, Talk with adults AUG 24th, Talk with Experts(Biologists)

Human Practice

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THANK YOU