A Biosensor Toolkit for Profiling Aromatics in Environment Peking iGEM 2013
C O O H COOH O H O H CH 3 C l C l N H 2 C O O H C l O H 2
Expensive Instrument Specialized Skills Confined in the lab 3
Inexpensive Alternative Techniques Accessible to the public Biosensor Toolkit In-field application 4
Advanced Biosensors Equipment XylS Adaptor NahR DmpR Biosensor Band-pass Filter Toolkit HbpR XylR HcaR 5
Biosensors C O O H COOH C O O H O H XylS X,R C O O H C O O H C O O H O C O C H O H O H 3 NahR O H O H O H OH DmpR OH X , R HbpR H O H N 2 XylR CH 3 CH 3 COOH HcaR Cl CH 3 6
Advanced Equipment Undetectable Aromatic Compounds Adaptor Adaptor Biosensor Biosensor Band-pass Filter 7
Advanced Advanced Signal Equipment Equipment Adaptor Adaptor Band-pass Filter Adaptor Conc. Low Medium High Band-pass Filter Conc. Conc. Conc. 8
Advanced Biosensors Equipment XylS Adaptor NahR DmpR Band-pass Filter HbpR XylR HcaR 9
To Profile Aromatics in Practical Samples A Collection of Biosensors for : C H 3 Aromatic Hydrocarbons C l C O O H Aromatic Acids A Bioinformatics O H Mining Method O H Phenols H O Biphenyls 10
TRs in Genetic-Context-Clear bacteria : Criterion 1 Criterion 1 Criterion 1 Criterion 1 Criterion 1 E. coli, B. subtilis, P. putida Transcriptional Regulators = TRs Aromatics -related TRs: Criterion 2 Criterion 2 Criterion 2 Criterion 2 Criterion 2 Keywords : aromatic, benzene, phenyl etc. Well studied TRs : Criterion 3 Criterion 3 Criterion 3 Criterion 3 Criterion 3 Score the TFs by their citations 11
Efficient Effective fea- 17 Manual Adjustment sible Promising Candidates sensors Excluding false positive cases Well 60 Studied TRs Criterion 3 Aromatics- 912 related TRs Criterion 2 TRs in genetic- 21,096 context-clearbacteria Criterion 1 4 3 , 3 6 2 , 5 3 7 12
Protein Regulated Sources Reported Typical Inducers Names Promoter 1 XylS Pm Pseudomonas putida (Arthrobacter siderocapsulatus) benzoic acid 2 XylR Pu Pseudomonas putida (Arthrobacter siderocapsulatus) m-Xylene tyrR Escherichia coli (strain K12) tyrosine 3 nahR Psal Pseudomonas putida (Arthrobacter siderocapsulatus) salicylic acid CapR Pseudomonas putida (Arthrobacter siderocapsulatus) phenol 4 hcaR Pc Escherichia coli (strain K12) 3-Phenyl-propionic acid 5 phenol dmpR Po Pseudomonas sp. (strain CF600). pobR Pseudomonas putida (Arthrobacter siderocapsulatus) p-hydroxybenzoic acid CymR Pseudomonas putida (Arthrobacter siderocapsulatus) 4-Isopropyl benzoate 6 PaaX P z Escherichia coli (strain K12) phenylacedtyl-CoA 7 (3-hydroxy-phenyl)-acetic acid hpaR P G Pseudomonas putida (Arthrobacter siderocapsulatus) mhpR Escherichia coli (strain K12) (3-hydroxy-phenyl)-propionic acid phhR Pseudomonas putida (Arthrobacter siderocapsulatus) phenylalanine 2-hydroxy-6-oxo-6-phenylhexa-2,4- bphS Pseudomonas sp. (strain CF600). dienoic acid 8 HbpR P C Pseudomonas nitroreducens 2-hydroxybiphenyl phcR Pseudomonas putida (Arthrobacter siderocapsulatus) phenol yodB Bacillus subtilis (strain 168) 2-methyl hydroquinone 13
Circuit Frame for Biosensors TR sfGFP sfGFP Constitutive Inducible promoter promoter pSB4K5 pUC57 14
Inducer Concentration Gradient NahR XylS Inducer Concentration Gradient HcaR DmpR HpaR HbpR PaaX XylR 15
Round 1 Round 2 Fine-tune TR Expression Fine-tune Reporter Amplitude J23113 J23109 B0034 J23117 sfGFP TR B0032 J23114 B0031 J23105 B0033 J23106 Constitutive Promoters Ribosome Binding Sites 16
Round 1: Fine-tuning HbpR expression level Best Optimized Original Induction Ratio Induced Fluorescence = Basal Fluorescence Constitutive promoter strength 17
Round 2: Fine-tuning sfGFP expression amplitude RBS strength Best Optimized Original 18
HbpR NahR Fine-tuning XylR XylS NahR DmpR HcaR XylS HcaR HpaR DmpR HpaR HbpR PaaX PaaX XylR 19
CH 3 H N H O COOH 2 OH CH 3 OH Cl CH 3 X,R COOH OH OH COOH OCOCH 3 C O O H COOH O H OH R R 2 2 C O O H R R 1 1 COOH C O O H OH O H COOH COOH C O O H X , R X,R HOOC X,R XylR 20 H O O C X , R X,R
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Practical Analysis Biosensor Characterization Synergistic Inducer Biosensor Biosensor Inducer Orthogonal Irrelevant Aromatics Antagonistic 22
Data Plot Orthogonality Assay Biosensor Synergistic Y-axis: A+ B+ Slope=1 Inducer Irrelevant X-axis Aromatics A+ B- Regression Line Orthogonal Y-axis A+ B+ Antagonistic X-axis: A+ B- 23
Salicylates Biphenyls Benzoates DmpR: Phenols Phenols Salicylates Biphenyls XylS: Benzoates 24
Phenols Benzoates Biphenyls NahR: Salicylates Salicylates Phenols Benzoates HbpR: Biphenyls 25
26
No Synergistic or A Large Antagonistic effects set of aromatics biosensors Practical Analysis 27
Fluorescence Intensity Fluorescence Output Water Sample Calibration Curve Lg( Conc.) Measured Conc. Biosensor Evaluating Reliability XylS NahR DmpR Measured Concentration Deviation = Real Concentration Fold HbpR XylR HcaR <<1 ≈ 1 >>1 Good Bad Bad 28
3-MeBzO 4-MeSaA NahR XylS HbpR 2-HBP Phl Interference 29
3-MeBzO 0 μM 3-MeBzO 10 μM 3-MeBzO 100 μM Deviation 4-MeSaA 4-MeSaA 4-MeSaA 4-MeSaA 4-MeSaA 4-MeSaA 4-MeSaA 4-MeSaA 4-MeSaA 0 μM 1 μM 10 μM 0 μM 1 μM 10 μM 10 μM 0 μM 1 μM Fold 0 μM Phl 2-HBP 0 μM 4.0 10 μM Phl 100 μM 2.5 93.4% Phl 0 μM 2-HBP 10 μM Phl 2.0 Fold between 10 μM Phl 1.0 100 μM 0.5 and 2.0 Phl 0.5 0 μM Phl 2-HBP 100 μM 10 μM 0.4 Phl 100 μM Phl 30 0.25
Band-pass Filter Band-pass Filter Adaptor Expanding The Detection Profile User-friendly Display 31
Expand The Detection Profile? OH CHO Adaptor Biosensor Biosensor NahF OH Convert the Undetectable NahR into the Detectable! COOH 32
NahF Adaptor Constitutive promoter O H O H C O O H C H O OH Biosensor COOH NahR sfGFP sfGFP Psal Pr 33
Induction Ratio Induced Fluorescence = Basal Fluorescence 34
Adaptor CHO C O O H CH 3 C H 3 Biosensor 35
C H O H 2 Adaptor 2 Adaptor 1 C H 3 XylB C H O H CHO C O O H 2 C H O C H O C H CH 3 3 C H C H C H 3 3 3 Biosensor XylC C O O H XylS C H 3 36
Convert the Undetectable Adaptor_XylB into the Detectable! Alcohol Aldehyde Adaptor_XylC Adaptor_NahF Aldehyde Acid Aldehyde Acid Sensor_XylS Sensor_NahR Sensing Acid Sensing Acid 37
Band-pass Filter Adaptor Expanding The Detection Profile User-friendly Display 38
Strain Strain Strain Output 1 2 3 Strain 1 Strain 2 Strain 3 Conc.1 Conc.2 Conc.3 Conc.4 Concentration 39
Positive Loop Negative Loop Output Output Input Input Input K 2 K 1 Output Output Incoherent Feed-forward Loop Input K 2 K 1 40
Mathematical Analysis Most Robust 41
Low High Hill Dissociation Coefficient Constant cI B C ϕ R73 δ 42
Positive Loop (Fixed IPTG) SaA IPTG SaA IPTG Ptac Psal Hybrid Promoter Negative Loop (Fixed SaA) Hybrid Promoter P2 F Promoter cI cI ϕ R73 δ ϕ R73 δ binding site -35 element -10 element CI binding site OR1 43
Determining The Design Frame Selecting Appropriate Proteins Engineering The Hybrid-promoter Characterizing The Hybrid-promoter Final Assembly 44
Band-pass Filter Adaptor Expanding The Detection Profile User-friendly Display 45
Achievements Future Plan To accomplish Band-pass 6 High-quality Biosensors Filter Construction 2 Adaptors More Adaptors Band-pass Filter 45 Biobricks Submitted 46
Pollution Reporters Monitoring Biosensors Degradation Bioremediation Pathways 47
Aromatics-sensing Regulators for Metabolic Process Control 48
Visiting a Printing Factory 49
Questionnaires 50
1.What are Aromatic Compounds? Compounds with heavy metal ion Fragrant Compounds Benzene Series Compounds 51
6.What do you think of biological prevention? No idea Totally for it Acceptable Totally Against it 52
Sow Synthetic Biologist for the future Providing Guidance to a High School iGEM Team 53
54 Team Communication
Víctor de Lorenzo Victoria Shingler Jan Roelof van der Meer 55
• 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 56
Thank all the team members 57
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