CAM BOY NJAU_China CONTENTS Syste Sy stem De Desig sign Mode - - PowerPoint PPT Presentation

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CAM BOY NJAU_China CONTENTS Syste Sy stem De Desig sign Mode - - PowerPoint PPT Presentation

Apr 16, 2023 104 likes •656 views

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

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CAM BOY

NJAU_China

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CONTENTS

Sy Syste stem De Desig sign Motiv tivatio tion& Bac & Background nd Wet L t Lab ab Mode

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Prac actic tice

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How Howeve ever… r… Mo Motiv ivat atio ion

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CONTENTS

Sy Syste stem De Desig sign Motiv tivatio tion& Bac & Background nd Wet L t Lab ab Mode

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Prac actic tice

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System Overview Sys ystem Design ign

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Tasks for CAM BOY

Sensitive enough to save lives！ Clean out pollution environmental friendly. Efficiency is important! Make it come true!

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Fundamental System Fundame undament ntal al Sys ystem

PcopA: Copper Sensor T7 Polymerase: Specific activation Gshf: GSH synthesis

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Sensor Choice

E.Coli genome—marO regulon

Fundame undament ntal al Sys ystem

Hao, Z., Lou, H., Zhu, R., Zhu, J., Zhang, D., Zhao, B. S., ... & Chen, P. R. (2013).

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Sensor Choice

cue (Cu efflux) system-- copA and cueO Now we’ve got three candidates for our sensor!

Fundame undament ntal al Sys ystem

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GSH can cope with copper pollution!

Glutathione (γ-glutamyl-cysteinyl-glycine; GSH)

Fundame undament ntal al Sys ystem

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Liu J. (2012).

Fundame undament ntal al Sys ystem

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gshF--Bifunctional enzyme

  • gshF: γ--GCS-GS
  • Streptococcus thermophiles

Fundame undament ntal al Sys ystem

Janowiak, B. E. & Griffith, O. W. (2005).

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Tasks for CAM BOY

Sensitive enough to save lives！ Clean out pollution environmental friendly. Efficiency is important! Make it come true!

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Facilitating System

Pvgb: Oxygen sensitive promoter dCas9: Repress Cu(Ⅰ) efflux pump

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Pvgb

Because… So…

Here’s for help! Can be maximally induced under microanaerobic conditions!

Fac acilit ilitat ating ing Sys ystem

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CRISPRi system Fac acilit ilitat ating ing Sys ystem

20 nt: specific DNA binding 42 nt: hairpin for Cas9 binding 40 nt transcription terminator

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Silencing copper efflux gene!

Copper efflux pump coding gene -- CopA

Fac acilit ilitat ating ing Sys ystem

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Tasks for CAM BOY

Sensitive enough to save lives！ Clean out pollution environmental friendly. Efficiency is important! Make it come true!

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Device Model for CAM BOY

  • A model on the process of Cu2+ elimination.
  • Also would be our future work!

Devi Device ce Mo Model

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Tasks for CAM BOY

Sensitive enough to save lives！ Clean out pollution environmental friendly. Efficiency is important! Make it come true!

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CONTENTS

Sy Syste stem De Desig sign Motiv tivatio tion& Bac & Background nd Wet L t Lab ab Mode

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Prac actic tice

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Wet Lab

Fundamental System Experiments Facilitating System Experiments

  • Sens

ensor

  • r
  • GSH and

and gs gshF

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Fundamental System Experiment

sensitive specific

pcopA maro pcueO

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pcueO+gfp maro+gfp pcopA+gfp

1mg/L copper for 2h

50 100 150 200 250 300 20 40 60 80 100 120 140 160 Relative Fluorescence Intensity(%of control) Time（min）

0.25mg/L 0.5mg/L 1mg/L 2mg/L 4mg/L 8mg/L

50 100 150 200 250 300 20 40 60 80 100 120 140 160 Relative Fluorescence Intensity(%of control) Time(min) 50 100 150 200 250 300 20 40 60 80 100 120 140 160 Relative Fluorescence Intensity(%of control) Time(min)

Sens nsit itiv ivit ityExperime iment nt

RFI=experiment group FI/control group FI

（relative fluorescence intensity）

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Speci Specifi fici city tyExperime iment nt

50 100 150 200 250 300 350 3 6

Relative Fluorescence Intensity(%of control) Time(h)

Al Li Mn Pb Zn Cu

pcueO+gfp

50 100 150 200 250 300 350 3 6

Relative Fluorescence Intensity(%of control) Time(h)

Al Li Mn Pb Zn Cu

maro+gfp pcopA+gfp

50 100 150 200 250 300 350 3 6

Relative Fluorescence Intensity(%of control) Time(h)

Al Li Mn Pb Zn Cu

4mg/L each 3h

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4mg/L copper for 5h

copper

pcopA RBS T7 polymerase

GFP reporter

pcopA RBS T7 polymerase

CueR

Fundame undament ntal E al Experime iment nt

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Will GSH detoxify copper in water ?

Fundame undament ntal E al Experime iment nt

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Fundame undament ntal E al Experime iment nt

Chlamydomonas reinhardtii

GSH is a ideal detoxicant for copper !

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XbaI XbaI SpeI

Fundame undament ntal E al Experime iment nt

T7 RBS gshf

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Wet Lab

Fundamental System Experiments Facilitating System Experiments

  • Pvgb
  • CRISPRi
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Pvgb Fac acilit ilitat ating ing E Experim iment nt

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CRISPRi

0.02 0.04 0.06 0.08 0.1 0.12 120 240 360 480

DH5α+Tet in 200ppm dcas9+Tet in 200ppm dcas9-Tet in 200ppm

p（tetR）

RBS

dcas9

J23100

SgRNA aims at copA

Fac acilit ilitat ating ing E Experim iment nt

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CONTENTS

Sy Syste stem De Desig sign Motiv tivatio tion& Bac & Background nd Wet L t Lab ab Mode

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Prac actic tice

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Int Introduc uctio ion

Temperature Sensor Skeleton Polymer membrane Heater

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Mo Model P l Procedur dure

Model Analysis Assumption Equations Derivation Simulation Sensitivity Analysis

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Mo Model Analys l Analysis is

 How do Cu2+ enter this device?  How do E.coli assimilate Cu2+ ?  How do E.coli grow?

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Assu ssumpti ption

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 The growth of E.coli is not influenced by Cu2+  The growth of E.coli is subject to logistic model  Cu2+ uptaking rate is constant  Cu2+ pass the polymers membrane by simple diffusion  Uniformity of Cu2+ and E.coli

150mg/L 152.5mg/L 155mg/L 157.5mg/L 160 mg/L DH5α + + + + + BL21 + + + + +

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Mo Model D l Develo lopme ment nt

j i

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Sim imulat ulatio ion

Figure 2 Simulation of the Cu2+ uptakenProcess with R = 0.5, h = 1, k = 0.075, λ = 0.0005, r = 0.1, K = 30000, D = 1/3000. Initial Cu2+ outside is 1mg/L and inside is 0. Figure 3 Growth curve of E.coli, with a initial concentration of 100CFU/L.

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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.

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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.

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Sa Safet fety

Protective Screen ≤ 0.22μm

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CONTENTS

Sy Syste stem De Desig sign Motiv tivatio tion& Bac & Background nd Wet L t Lab ab Mode

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Prac actic tice

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Worksho rkshop

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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

Quest stio ionnai naire re Survey vey

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Exhibit hibition ion

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Exhibit hibition ion

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Commu mmunic icat ation ion

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Collabor llaborat ation ion

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Abou About B BUGs

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 Prof. Jiang Jiandong  Prof. Yan Xin  Zhang Long  Chen Xueting  Zou Shenshen  All team members

Sponsors

Acknowled nowledgement gement

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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

  • utsourcing 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.

Acknowled nowledgement gement

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Welcome questions and suggestions!