A Biosensor Toolkit for Profiling Aromatics in Environment Peking - - PowerPoint PPT Presentation

A Biosensor Toolkit for Profiling Aromatics in Environment Peking iGEM 2013

COOH CH3

O H C l C l C l

O H

C O O H

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Expensive Instrument Confined in the lab Specialized Skills

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Biosensor Toolkit

Alternative Techniques

Inexpensive In-field application Accessible to the public

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Biosensors Advanced Equipment XylS NahR DmpR HbpR XylR HcaR Adaptor Band-pass Filter Biosensor Toolkit

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Biosensors XylS NahR DmpR HbpR XylR HcaR

COOH X,R

C O O H

C O O H O H C O O H O H

C O O H O H O H

C O O H O C O C H

3

O H

OH OH

O H X , R

H O

H

2

N

COOH

CH3 Cl

CH3 CH3

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Adaptor Advanced Equipment Biosensor

Undetectable Aromatic Compounds

Band-pass Filter Adaptor Biosensor

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Advanced Equipment Band-pass Filter Adaptor Adaptor Advanced Equipment Band-pass Filter Adaptor

Low Conc. Medium Conc.

Conc. Signal

High Conc.

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Biosensors XylS NahR DmpR HbpR XylR HcaR Advanced Equipment Adaptor Band-pass Filter

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To Profile Aromatics in Practical Samples

A Collection of Biosensors for :

Aromatic Acids Phenols Biphenyls Aromatic Hydrocarbons

C H

3

C l

C O O H O H

O H

H O

A Bioinformatics Mining Method

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Transcriptional Regulators = TRs Criterion 1 Criterion 2 Criterion 3 Criterion 1 Criterion 2 Criterion 3 TRs in Genetic-Context-Clearbacteria:

• E. coli, B. subtilis, P. putida

Criterion 1 Criterion 2 Criterion 3 Aromatics-related TRs:

Keywords: aromatic, benzene, phenyl etc.

Criterion 1 Criterion 2 Criterion 3 Well studied TRs:

Score the TFs by their citations

Criterion 1 Criterion 2 Criterion 3

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TRs in genetic- context-clearbacteria Aromatics- related TRs fea- sible sensors Well Studied TRs

Criterion 3 Criterion 2 Criterion 1 4 3 , 3 6 2 , 5 3 7 21,096 912 60

Effective Efficient

Excluding false positive cases

Manual Adjustment 17

Promising Candidates

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8 6 4 3 2 1 5 7

Protein Names Regulated Promoter Sources Reported Typical Inducers

XylS Pm Pseudomonas putida (Arthrobacter siderocapsulatus) benzoic acid XylR Pu Pseudomonas putida (Arthrobacter siderocapsulatus) m-Xylene tyrR Escherichia coli (strain K12) tyrosine nahR Psal Pseudomonas putida (Arthrobacter siderocapsulatus) salicylic acid CapR Pseudomonas putida (Arthrobacter siderocapsulatus) phenol hcaR Pc Escherichia coli (strain K12) 3-Phenyl-propionic acid dmpR Po Pseudomonas sp. (strain CF600). phenol pobR Pseudomonas putida (Arthrobacter siderocapsulatus) p-hydroxybenzoic acid CymR Pseudomonas putida (Arthrobacter siderocapsulatus) 4-Isopropyl benzoate PaaX Pz Escherichia coli (strain K12) phenylacedtyl-CoA hpaR PG Pseudomonas putida (Arthrobacter siderocapsulatus) (3-hydroxy-phenyl)-acetic acid mhpR Escherichia coli (strain K12) (3-hydroxy-phenyl)-propionic acid phhR Pseudomonas putida (Arthrobacter siderocapsulatus) phenylalanine bphS Pseudomonas sp. (strain CF600). 2-hydroxy-6-oxo-6-phenylhexa-2,4- dienoic acid HbpR PC Pseudomonas nitroreducens 2-hydroxybiphenyl phcR Pseudomonas putida (Arthrobacter siderocapsulatus) phenol yodB Bacillus subtilis (strain 168) 2-methyl hydroquinone

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TR

Constitutive promoter

sfGFP

Inducible promoter

Circuit Frame for Biosensors

pSB4K5 pUC57

sfGFP

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XylS HbpR XylR DmpR HcaR HpaR PaaX NahR Inducer Concentration Gradient Inducer Concentration Gradient

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TR

J23113 J23109 J23117 J23114 J23105 J23106 B0034 B0032 B0031 B0033 sfGFP Fine-tune TR Expression

Round 1

Fine-tune Reporter Amplitude

Round 2

Constitutive Promoters Ribosome Binding Sites

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Constitutive promoter strength

Round 1: Fine-tuning HbpR expression level

Induction Ratio =

Induced Fluorescence Basal Fluorescence

Best Optimized Original

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RBS strength

Round 2: Fine-tuning sfGFP expression amplitude

Best Optimized Original

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Fine-tuning

XylS HbpR XylR DmpR HcaR HpaR PaaX NahR XylS HbpR XylR DmpR HcaR HpaR PaaX NahR

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COOH X,R

C O O H O H R

1

R

2

C O O H

C O O H X , R C O O H O H

H O O C X , R

COOH OH OH COOH OCOCH3 COOH OH R1 R2 COOH OH COOH X,R HOOC

X,R

OH OH X,R

XylR

H O

H

2

N

CH3 Cl CH3 CH3 COOH

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Biosensor Characterization Practical Analysis

Synergistic Antagonistic Orthogonal

Inducer Biosensor Irrelevant Aromatics Inducer Biosensor

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Orthogonality Assay Data Plot

X-axis X-axis: A+ B- Y-axis Y-axis: A+ B+ Regression Line Orthogonal Slope=1 Synergistic Antagonistic

Biosensor

A+ B- A+ B+

Inducer Irrelevant Aromatics

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Benzoates Salicylates Biphenyls DmpR: Phenols Salicylates Biphenyls Phenols XylS: Benzoates

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Benzoates Salicylates Phenols HbpR: Biphenyls Benzoates Biphenyls Phenols NahR: Salicylates

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A Large set of aromatics biosensors No Synergistic or Antagonistic effects

Practical Analysis

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Water Sample Biosensor

XylS NahR DmpR HbpR XylR HcaR Calibration Curve

Lg( Conc.) Fluorescence Intensity Fluorescence Output Measured Conc.

Measured Concentration Real Concentration Deviation Fold Evaluating Reliability

<<1 Bad ≈1 Good >>1 Bad

=

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XylS NahR HbpR

Interference

3-MeBzO 4-MeSaA 2-HBP Phl

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4.0 2.5 2.0 1.0 0.5 0.4 0.25 Deviation Fold

93.4% Fold between 0.5 and 2.0

3-MeBzO 0 μM 3-MeBzO 10 μM 3-MeBzO 100 μM

4-MeSaA

0 μM

4-MeSaA

0 μM

4-MeSaA

0 μM

4-MeSaA

1 μM

4-MeSaA

1 μM

4-MeSaA

1 μM

4-MeSaA

10 μM

4-MeSaA

10 μM

4-MeSaA

10 μM

2-HBP 0 μM 2-HBP 10 μM 2-HBP 100 μM Phl

0 μM

Phl

0 μM

Phl

0 μM

Phl

10 μM

Phl

10 μM

Phl

10 μM

Phl

100 μM

Phl

100 μM

Phl

100 μM

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Expanding The Detection Profile User-friendly Display

Adaptor

Band-pass Filter Band-pass Filter

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Biosensor

Adaptor

Biosensor

Expand The Detection Profile? Convert the Undetectable into the Detectable!

OH CHO

OH COOH

NahF NahR

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Psal Pr sfGFP NahR NahF

Constitutive promoter

sfGFP

OH COOH

Adaptor Biosensor

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Induction Ratio =

Induced Fluorescence Basal Fluorescence

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CHO CH3

C O O H C H

3

Adaptor Biosensor

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Adaptor 2 Adaptor 1

C H

2

O H C H

3

C H O C H

3

CHO CH3

C O O H C H

3

Biosensor

XylC XylS XylB

C H

2

O H C H

3

C H O C H

3

C O O H C H

3

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Adaptor_XylB

Alcohol Aldehyde

Adaptor_NahF

Aldehyde  Acid

Adaptor_XylC

Aldehyde Acid

Sensor_XylS

Sensing Acid

Sensor_NahR

Sensing Acid

Convert the Undetectable into the Detectable!

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Expanding The Detection Profile

Band-pass Filter

Adaptor

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User-friendly Display

Conc.1 Conc.2 Conc.3 Conc.4

Strain 1 Strain 2 Strain 3 Strain 1 Strain 2 Strain 3 Concentration Output

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Input Output

Incoherent Feed-forward Loop Output Input

K2 K1

Input Output

K1

Positive Loop

Input Output

K2

Negative Loop

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Mathematical Analysis Most Robust

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B

cI

ϕR73δ

C

High Hill Coefficient Low Dissociation Constant

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IPTG SaA

ϕR73δ

• 10 element

ϕR73δ binding site -35 element CI binding site OR1

cI cI

IPTG SaA

Ptac Psal

Positive Loop (Fixed IPTG) Negative Loop (Fixed SaA)

Hybrid Promoter P2 F Promoter Hybrid Promoter

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Determining The Design Frame Selecting Appropriate Proteins Engineering The Hybrid-promoter Characterizing The Hybrid-promoter Final Assembly

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Expanding The Detection Profile

Band-pass Filter

Adaptor

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User-friendly Display

Achievements

6 High-quality Biosensors 2 Adaptors Band-pass Filter 45 Biobricks Submitted

Future Plan

To accomplish Band-pass Filter Construction More Adaptors

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Biosensors

Reporters Degradation Pathways Pollution Monitoring Bioremediation

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Aromatics-sensing Regulators for Metabolic Process Control

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Visiting a Printing Factory

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Questionnaires

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1.What are Aromatic Compounds?

Compounds with heavy metal ion Fragrant Compounds Benzene Series Compounds

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6.What do you think of biological prevention?

No idea Totally for it Acceptable Totally Against it

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Providing Guidance to a High School iGEM Team Sow Synthetic Biologist for the future

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Team Communication

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Victoria Shingler Víctor de Lorenzo Jan Roelof van der Meer

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• Dr. Chunbo Lou
• Prof. Qi Ouyang
• Prof. Luhua Lai
• Prof. Zhen Yang
• Prof. Junfeng Hu
• Prof. Chongren Xu
• Prof. Xinqiang He
• Prof. Chunxiong Luo
• Prof. Ge Gao
• Dr. Zailing Bai

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Thank all the team members

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