University of Wisconsin - Madison igem Team 2012
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1 University of Wisconsin - Madison igem Team 2012
2 Translational Coupling: A tool for evaluating the translation of heterologous proteins in Escherichia coli Troubleshooting a limonene production pathway in E. coli
3 1 Let s do Some Cloning!
4 2 Let s do Some Cloning!
5 3 Let s do Some Cloning!
6 4 Let s do Some Cloning!
7 5 Applications of Limonene
8 Limonene as a Biofuel Standards: Jet A (A-1) Standards Jet A* Ethanol Dodecane Limonene Flash point ( C) Min: Autoignition temp ( C) Freezing Point ( C) Max: 40 ( 47) Boiling Point ( C) Max: 300 (340) C (kg/l) ** Heat of Comb. (MJ/kg) Min: Energy density (MJ/L) Notes: * British Petroleum; ** Density@20 C; 6
9 Enzymatic Production of Limonene Acetyl-CoA HMG Mevalonate IPP Mevalonate 5 Phosphate GPP Limonene FPP DMAPP 7 Martin et al. Nat. Biotech Amorphadiene
10 Production Vectors Acetyl-CoA HMG pbba5c Mevalonate IPP Mevalonate 5 Phosphate Acetyl-CoA OVERALL GPP FPP Limonene FPP DMAPP 8 Amorphadiene
11 Production Vectors pbba5c with geranyl pyrophosphate pbba5c synthase insertion Acetyl-CoA HMG Mevalonate IPP Mevalonate 5 Phosphate Acetyl-CoA OVERALL GPP FPP GPP LIMONENE! FPP DMAPP 9 Amorphadiene
12 Production Vectors Acetyl-CoA HMG J23102 Limonene synthase J23102-BBa_I Mevalonate IPP J23102 Mevalonate 5 Phosphate GPP LIMONENE! FPP DMAPP 10 Amorphadiene
13 Production Vectors Acetyl-CoA HMG Amorphadiene synthase Mevalonate IPP Mevalonate 5 Phosphate GPP LIMONENE! FPP DMAPP 11 Amorphadiene
14 Summary of Production Strains pbba5c Acetyl-Coa HMG Mevalonate + Amorphadiene IPP synthase + J23102 Limonene synthase Mevalonate 5 Phosphate GPP OR J23102 LIMONENE! = = FPP 12 DMAPP Amorphadiene Limonene Amorphadiene
15 Summary of Production Strains pbba5c with geranyl pyrophosphate synthase insertion Acetyl-Coa HMG Mevalonate IPP + J23102 Limonene synthase Mevalonate 5 Phosphate GPP J23102 = LIMONENE! FPP 13 DMAPP Limonene Amorphadiene
16 Production Assay Diluted Extracted Diluted 5 ml overnight cultures 40 ml cultures IPTG induced Dodecane overlay Gas chromatography Mass spectrometry 14
17 Total Ion Count Total Ion Count Production Assay Results Total Ion Count (X10,000,000) (X10,000) (X10,000) Target Compound (Limonene Standard) Limonene pbba5c + pads pbba5c-gpps + pads (X10,000) Library Standard GC-MS Similarity Search Results pbba5c + J Dodecane pbba5c + J23102-LIMS Retention Time (min) Retention Time (min) 15
18 Dissecting the Problem pbba5c Acetyl-Coa HMG Mevalonate IPP J23102 Limonene synthase Amorphadiene synthase Mevalonate 5 Phosphate GPP + Where do we start? J23102 = LIMONENE! FPP 17 DMAPP Limonene Amorphadiene Amorphadiene
19 Central Dogma Step: DNA mrna Protein Phenotype Confirmation: Sequencing J23102 well Questionable Nothing characterized 18
20 Percentage of Codons Codon Usage in E. Coli Classic LimS1 30 CO LimS1 Native MalE Codon Quality 19
21 Troubleshooting Translation Pros Method Cons Specific Quantifiable Mass Spectrometry Expensive Multi-variable Functionality unknown Sensitive Quantifiable Western Blot Expensive Functionality unknown Protein functionality Enzymatic activity In vitro assay Purification purification Tags may alter activity 20
22 Translational Coupling Cassette Ribosome The When Liberated stop Reporter codon your helicase RBS gene releases gene is activity is availible being is the translated melts fully ribosome for the translated binding hairpin 21
23 Translational Coupling Cassette The RBS is If the sequestered target gene and the is not reporter translated gene is not translated 22
24 Cloning Into the TCC Is it translating? Translation? Gene of Interest Reporter Gene (RFP) 23
25 Translational Coupling Cassette ` 24 Mendez-Perez et al. Metab Eng 2012
26 Characterization of the Cassette Two genes were cloned into the cassette for characterization E0040: green fluorescent protein Positive control Limonene synthase with an embedded stop codon Negative control GFP-TCC-RFP LimS1-Stop-TCC 25
27 96-Well Plate Reader Assay Diluted 5 ml overnight cultures 96 well plate Tecan Fluorescence plate reader 26
28 96-well Characterization Data Strain LimS1(NS) TCC-RFP 12 Hours 18 Hours COLimS1(MS) 24 Hours COLimS1(NS) GFP-TCC-RFP RFP / OD 600 (au) 27
29 Typhoon Imager Assay Dropped culture Incubated overnight 5 ml overnight cultures Drop plate Scanned in typhoon imager 28
30 Typhoon Plate Reader Data GFP-TCC-RFP TCC-RFP LimS1-TCC LimS1-Stop-TCC COLimS1-TCC COLimS1-Stop-TCC
31 Typhoon Plate Reader Data GFP-TCC-RFP TCC-RFP LimS1-TCC LimS1-Stop-TCC COLimS1-TCC COLimS1-Stop-TCC
32 Strains Typhoon Plate Reader Data Typhoon Pixel Density 29
33 Conclusions from Translational Coupling Cassette Limonene synthase (Bba_I742111) is not being translated Translation of codon-optimized limonene synthase The cassette works as designed Available for all teams to use (BBa_K762000) 30
34 Total Ion Count Further Production Assays pbba5c with geranyl pyrophosphate pbba5c synthase insertion (X10,000,000) Limonene pbba5c-gpps + J23102-COLIMS1 + pbba5c-gpps + J J23102 Dodecane Dodecane Retention Time (min) 31
35 Conclusions and Future Steps pbba5c with geranyl pyrophosphate synthase insertion Acetyl-Coa HMG Mevalonate IPP In vitro assay after purification Western blot with His-Tag GPP antibodies Different production techniques FPP J J23102 Limonene synthase = Clone into TCC Mevalonate 5 Phosphate Run in vitro assay after purification LIMONENE! Western blot with His- Tag antibodies 32 DMAPP Limonene Amorphadiene
36 Translational Coupling Cassette A synthetic biology tool for troubleshooting the expression of heterologous proteins Gene Target of Interest Gene Reporter Gene
37 33 igem Outreach
38 igem Outreach James Madison Memorial High School advanced placement biology classes 34
39 Lab Visits and Brainstorming Sessions Facilitating the formation of their high school igem team 35
40 Acknowledgements Special thank you to: Brian Pfleger PhD Matthew Copeland PhD Jennifer Reed PhD Sophia Weerth UW-Madison Biochemistry Dept. UW-Madison Engineering Dept. 36
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