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

Background � B reeding bio - insecurity： 2001 Anthrax Attacks � Leaked from a biology lab �

Background � Intellectual property rights �

How to prevent? � Moral ity or Laws � Biot echnology �

Inspiration � Abstract � Layered structure � Sequential control � Open � Error input protection � Settable password � Electronic circuit � Simple logic circuit �

Idea and Proposal Design � Layered AND Gates sRNA Input signals ： MIN System Inducers �

Layered AND Gates for Multiple Passwords [Ara] � araC � ipgC � pBAD � [IPTG] � pTac � lacI � pipaH* � sicA* � mxiE � AND gate: The transcription factors interact with chaperones to turn on the promoter of downstream �

Layered AND Gates for Multiple Passwords [Ara] � ipgC � araC � pBAD � [ IPTG ] � mxiE � pTac � pipaH* � sicA* � lacI � [aTC] � tetR � invF � pTet � psicA � From AND gate to layered AND gates �

Layered AND Gates for Multiple Passwords Three sensors have been assembled and their function has been verified using fluorescent protein as reporter. � AraC � [Ara] � araC � J23105 � B0034 � C0080 � B0010 � B0012 � K206000 � Control � Verification � LacI � [IPTG] � lacI � J23105 � B0034 � C0012 � B0012 � R0010 � B0010 � Control � Verification � TetR � [aTC] � tetR � J23105 � B0034 � C0040 � R0040 � B0010 � B0012 � Control � Verification �

Layered AND Gates for Multiple Passwords AND Gates were constructed successfully and verified through FACS and fluorescence microscope after 6 h induction. � Negative Ara and Ara � No IPTG � control � IPTG � induction �

sRNA Regulatory System for Sequential Control � Ribosome-bound mRNA � Expression � Ribosome � sRNA � mRNA � Repression � Hfq �

sRNA Regulatory System for Sequential Control � Regulation anti � MicC � plasmid � Target � anti � Lac Z � Lac Z � Target � target � O ne- step site-directed mutagenesis m e thod � Design of screening system for synthetic sRNA �

sRNA Regulatory System for Sequential Control � sRNA for ipgC � sRNA for sicA* � ipgC � sicA* � 38% � 66% � ipgC anti-ipgC � sicA * anti-sicA * � Using lacZ as reporter to determine the repressive efficiency of sRNAs

sRNA Regulatory System for Sequential Control � μ= ¡0.2 � μ= ¡2 � Stage(I) ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡Stage(II) � μ= ¡200 � μ= ¡20 �

MIN System for Quorum Control FtsZ � Cell division � MinCD MinC MinD inhibitor �

MIN System for Quorum Control Aii LuxR AHL � MIN R0063 � Design and construction of MIN system

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. �

Modeling and Prediction mxiE � MinC � ipgC � araC � araC � pBAD � pBAD � ipgC � ipgC � invF � sicA * � pipaH* � sicA* � pipaH* � sicA* � lacI � pTac � pTac � sRNA(ipgC) � sRNA(ipgC) � mxiE � mxiE � lacI � psicA � psicA � sRNA(MIN) � sRNA(MIN) � Aii � Aii � invF � invF � tetR � tetR � sRNA(sicA*) � sRNA(sicA*) � pTet � pTet � AHL � AHL � Right password � LuxR � LuxR � lux pL lux pL MIN � MIN � Turn on � Simulation of E. co -Lock

Modeling and Prediction mxiE � MinC � ipgC � invF � araC � araC � pBAD � pBAD � ipgC � ipgC � sicA * � sicA* � pipaH* � pipaH* � sicA* � lacI � pTac � pTac � sRNA(ipgc) � sRNA(ipgc) � mxiE � mxiE � lacI � psicA � psicA � sRNA(MIN) � sRNA(MIN) � Aii � Aii � pTet � pTet � sRNA(sicA) � sRNA(sicA) � invF � invF � tetR � tetR � AHL � AHL � Wrong password � Turn off � LuxR � LuxR � lux pL lux pL MIN � MIN � Simulation of E. co -Lock �

Applications � Biosafety � Ecological security � Bio-economic safety �

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 �

Human Practice 2 nd iGEM Tianjin COFCO � Conference � University � Beijing University of Chemical Technology �

Instructors & Team � Prof. Chun Li � Prof. Shuyuan Guo � A. Prof. Jun Li �