E. c coli li bas based lo lock Background Problems Bio-safety - - PowerPoint PPT Presentation

Eco Ecolo logic ically ally fr frie iendly ly, Eco Economic ically ally e effecti tive

• E. c

coli li bas based lo lock

Background

Problems

Bio-safety Intellectual property rights

Breeding bio-insecurity： 2001 Anthrax Attacks

Leaked from a biology lab

Background

Intellectual property rights

Background

Morality or Laws Biotechnology How to prevent?

Inspiration

Layered structure Sequential control Error input protection Settable password

Electronic circuit Abstract Simple logic circuit

Open

Idea and Proposal Design

Layered AND Gates Input signals： Inducers sRNA MIN System

AND gate: The transcription factors interact with chaperones to turn on the promoter of downstream

pBAD ipgC araC pTac mxiE lacI pipaH* sicA*

Layered AND Gates for Multiple Passwords

[IPTG] [Ara]

From AND gate to layered AND gates

pBAD ipgC araC

[Ara]

pTac mxiE lacI

[IPTG]

pTet invF tetR

[aTC]

pipaH* sicA* psicA

Layered AND Gates for Multiple Passwords

Three sensors have been assembled and their function has been verified using fluorescent protein as reporter.

Control Control Control Verification Verification Verification

K206000

[Ara]

J23105 B0034

araC

C0080 B0010 B0012

AraC

R0010 J23105 B0034 C0012 B0010 B0012

LacI lacI [IPTG]

R0040 J23105 B0034 C0040 B0010 B0012

TetR [aTC] tetR

Layered AND Gates for Multiple Passwords

AND Gates were constructed successfully and verified through FACS and fluorescence microscope after 6 h induction.

No induction Ara IPTG Ara and IPTG Negative control

Layered AND Gates for Multiple Passwords

sRNA Regulatory System for Sequential Control

Ribosome Ribosome-bound mRNA

Expression Repression

Hfq mRNA sRNA

Regulation plasmid

Design of screening system for synthetic sRNA

MicC Lac Z One-step site-directed mutagenesis method

Target

anti

Lac Z

anti

target

Target

sRNA Regulatory System for Sequential Control

Using lacZ as reporter to determine the repressive efficiency of sRNAs

sRNA for sicA* sRNA for ipgC

ipgC anti-ipgC sicA * anti-sicA *

sicA* ipgC 66% 38%

sRNA Regulatory System for Sequential Control

Stage(I) ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡Stage(II) μ= ¡0.2 μ= ¡2 μ= ¡20 μ= ¡200

sRNA Regulatory System for Sequential Control

MIN System for Quorum Control

Cell division

MinD MinC MinCD

inhibitor

FtsZ

Design and construction of MIN system

AHL

R0063

LuxR Aii MIN

MIN System for Quorum Control

Modeling of MIN system Simulation results:

Different initial AHL concentration for MIN system model. The higher value of AHL, the higher cell density.

MIN System for Quorum Control

ipgC araC pBAD pipaH* sicA* pTac mxiE lacI sRNA(ipgC) pTet invF tetR sRNA(sicA*) psicA sRNA(MIN) ipgC araC pBAD pTac mxiE lacI sRNA(ipgC) pipaH* sicA* pTet invF tetR sRNA(sicA*) psicA sRNA(MIN)

Simulation of E. co-Lock

Aii AHL lux pL MIN LuxR Aii AHL lux pL MIN LuxR

Modeling and Prediction

Right password

Turn on

MinC ipgC mxiE sicA* invF

ipgC araC pBAD pipaH* sicA* pTac mxiE lacI sRNA(ipgc) pTet invF tetR sRNA(sicA) psicA sRNA(MIN) ipgC araC pBAD pTac mxiE lacI sRNA(ipgc) pipaH* sicA* pTet invF tetR sRNA(sicA) psicA sRNA(MIN) Aii AHL lux pL MIN LuxR Aii AHL lux pL MIN LuxR

Modeling and Prediction

Simulation of E. co-Lock

Wrong password

Turn off

MinC ipgC mxiE sicA* invF

Applications

Biosafety Bio-economic safety Ecological security

Achievements

√ Design, construction, characterization and submission of 18 new biobrick parts and devices √ Collaboration with Tianjin University √ Construction of an approach (E. co-lock) to solve the problems of biosafety and biosecurity

Bio-security Bio-safety

Tianjin University

Beijing University

• f Chemical

Technology

COFCO

2nd iGEM Conference

Human Practice

Instructors & Team

• A. Prof. Jun Li
• Prof. Shuyuan Guo
• Prof. Chun Li