CAM BOY NJAU_China
CONTENTS Syste Sy stem De Desig sign Mode odel Motiv tivatio tion& Bac & Background nd Wet L t Lab ab Prac actic tice
Mo Motiv ivat atio ion How Howeve ever… r…
CONTENTS Syste Sy stem De Desig sign Mode odel Motiv tivatio tion& Bac & Background nd Wet L t Lab ab Prac actic tice
Sys ystem Design ign System Overview
Tasks for CAM BOY Sensitive enough to save lives ！ Clean out pollution environmental friendly. Efficiency is important! Make it come true!
Fundame undament ntal al Sys ystem Fundamental System P copA : Copper Sensor T7 Polymerase: Specific activation Gsh f: GSH synthesis
Fundame undament ntal al Sys ystem Sensor Choice Hao, Z., Lou, H., Zhu, R., Zhu, J., Zhang, D., Zhao, B. S., ... & Chen, P. R. (2013). E.Coli genome— marO regulon
Fundame undament ntal al Sys ystem Sensor Choice cue (Cu efflux) system-- copA and cueO Now we’ve got three candidates for our sensor!
Fundame undament ntal al Sys ystem GSH can cope with copper pollution! Glutathione (γ -glutamyl-cysteinyl-glycine; GSH)
Fundame undament ntal al Sys ystem Liu J. (2012).
Fundame undament ntal al Sys ystem gsh F--Bifunctional enzyme Janowiak, B. E. & Griffith, O. W. (2005). • gsh F : γ --GCS-GS • Streptococcus thermophiles
Tasks for CAM BOY Sensitive enough to save lives ！ Clean out pollution environmental friendly. Efficiency is important! Make it come true!
Facilitating System P vgb : Oxygen sensitive promoter dCas9: Repress Cu( Ⅰ ) efflux pump
Fac acilit ilitat ating ing Sys ystem P vgb Because… Can be maximally induced So… under microanaerobic conditions! Here’s for help!
Fac acilit ilitat ating ing Sys ystem CRISPRi system 20 nt: specific DNA binding 42 nt: hairpin for Cas9 binding 40 nt transcription terminator
Fac acilit ilitat ating ing Sys ystem Silencing copper efflux gene! Copper efflux pump coding gene -- Cop A
Tasks for CAM BOY Sensitive enough to save lives ！ Clean out pollution environmental friendly. Efficiency is important! Make it come true!
Devi Device ce Mo Model Device Model for CAM BOY • A model on the process of Cu 2+ elimination. • Also would be our future work!
Tasks for CAM BOY Sensitive enough to save lives ！ Clean out pollution environmental friendly. Efficiency is important! Make it come true!
CONTENTS Syste Sy stem De Desig sign Mode odel Motiv tivatio tion& Bac & Background nd Wet L t Lab ab Prac actic tice
Wet Lab Fundamental System Experiments • Sens ensor or • GSH and and gs gsh F Facilitating System Experiments
Fundamental System Experiment p copA sensitive maro specific p cueO
Sens nsit itiv ivit ityExperime iment nt 1 mg/L copper for 2 h RFI=experiment group FI /control group FI 300 Relative Fluorescence Intensity(%of control) （ relative fluorescence intensity ） 250 p cueO + gfp 200 150 100 50 0 0 20 40 60 80 100 120 140 160 Time(min) p copA + gfp 300 0.25mg/L Relative Fluorescence Intensity(%of control) 250 0.5mg/L 200 150 1mg/L 100 2mg/L 50 4mg/L 0 0 20 40 60 80 100 120 140 160 300 8mg/L Time （ min ） Relative Fluorescence maro + gfp Intensity(%of control) 250 200 150 100 50 0 0 20 40 60 80 100 120 140 160 Time(min)
Speci Specifi fici city tyExperime iment nt p copA +gfp 350 Relative Fluorescence Intensity(%of 300 Al 250 Li 4mg/L 200 Mn control) 150 each 3h Pb 100 Zn 50 Cu 0 0 3 6 p cueO + gfp maro + gfp Time(h) 350 350 Relative Fluorescence Intensity(%of control) Relative Fluorescence Intensity(%of control) 300 300 Al Al 250 Li 250 Li 200 Mn Mn 200 150 Pb Pb 150 100 Zn Zn 100 Cu 50 Cu 50 0 0 3 6 0 Time(h) 0 3 6 Time(h)
Fundame undament ntal E al Experime iment nt copper CueR p copA T7 polymerase RBS 4 mg/L copper for 5 h GFP p copA reporter T7 polymerase RBS
Fundame undament ntal E al Experime iment nt Will GSH detoxify copper in water ?
Fundame undament ntal E al Experime iment nt GSH is a ideal detoxicant for copper ! Chlamydomonas reinhardtii
Fundame undament ntal E al Experime iment nt T7 RBS gshf XbaI XbaI SpeI
Wet Lab Fundamental System Experiments Facilitating System Experiments • Pvgb • CRISPRi
Fac acilit ilitat ating ing E Experim iment nt P vgb
Fac acilit ilitat ating ing E Experim iment nt CRISPRi 0.12 DH5 α+ Tet 0.1 in 200ppm 0.08 dcas9+Tet in 200ppm 0.06 0.04 dcas9-Tet in 200ppm 0.02 0 120 240 360 480 J23100 p （ tetR ） dcas9 RBS SgRNA aims at copA
CONTENTS Syste Sy stem De Desig sign Mode odel Motiv tivatio tion& Bac & Background nd Wet L t Lab ab Prac actic tice
Int Introduc uctio ion Temperature Sensor Skeleton Polymer membrane Heater
Mo Model P l Procedur dure Model Analysis Sensitivity Assumption Analysis Equations Simulation Derivation
Mo Model Analys l Analysis is How do Cu 2+ enter this device? How do E.coli assimilate Cu 2+ ? How do E.coli grow?
Assu ssumpti ption on The growth of E.coli is not influenced by Cu 2+ 150mg/L 152.5mg/L 155mg/L 157.5mg/L 160 mg/L The growth of E.coli is subject to DH5 α + + + + + logistic model Cu 2+ uptaking rate is constant BL21 + + + + + Cu 2+ pass the polymers membrane by simple diffusion Uniformity of Cu 2+ and E.coli
Mo Model D l Develo lopme ment nt i j
Sim imulat ulatio ion Figure 3 Growth curve of E.coli, with Figure 2 Simulation of the Cu 2+ a initial concentration of 100CFU/L. uptakenProcess with R = 0.5, h = 1, k = 0.075, λ = 0.0005, r = 0.1, K = 30000, D = 1/3000. Initial Cu 2+ outside is 1mg/L and inside is 0.
Sens nsit itiv ivit ity Analys y Analysis is Figure 4. Morris SA. Mu represents the overall effect of the parameter on a given out put while Sigma means the higher-order characteristics of the parameter .
Sens nsit itiv ivit ity Analys y Analysis is Figure 4. Morris SA. Mu represents the overall effect of the parameter on a given out put while Sigma means the higher-order characteristics of the parameter .
Sa Safet fety ≤ 0.22μ m Protective Screen
CONTENTS Syste Sy stem De Desig sign Mode odel Motiv tivatio tion& Bac & Background nd Wet L t Lab ab Prac actic tice
Worksho rkshop
Quest stio ionnai naire re Survey vey Have you heard of synthetic biology before the survey? A 、 Yes B 、 No C 、 Not clear Have you heard of synthetic biology before the survey? A 、 Yes B 、 No C 、 Not clear
Exhibit hibition ion
Exhibit hibition ion
Commu mmunic icat ation ion
Collabor llaborat ation ion
Abou About B BUGs
Acknowled nowledgement gement Prof. Jiang Jiandong Sponsors Prof. Yan Xin Zhang Long Chen Xueting Zou Shenshen All team members
Acknowled nowledgement gement GENEWIZ, Inc. is a leading global genomics service company, including DNA sequencing, gene synthesis, molecular biology, genomic, bioinformatics, Clinical Services and GLP regulatory- compliant services. GENEWIZ leads the industry in outsourcing partnerships with pharmaceutical, biotechnology, clinical, academic, and government institutions. Found in 1999 and headquartered in South Plainfield, NJ, GENEWIZ, Inc. is a privately-held global enterprise with locations in Boston, MA; Washington, DC Metro; Research Triangle Park, NC; San Diego, CA; Los Angeles, CA; San Francisco, CA; and Seattle, WA. International locations include Beijing, China; Suzhou, China; London, United Kingdom; Langen, Germany; and Tokyo, Japan.
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