University of Wisconsin - Madison iGEM Team 2012 Translational - PowerPoint PPT Presentation

University of Wisconsin - Madison iGEM Team 2012

Translational Coupling : A tool for evaluating the translation of heterologous proteins in Escherichia coli Troubleshooting a limonene production pathway in E. coli

Let’s do Some Cloning ! 1

Let’s do Some Cloning ! 2

Let’s do Some Cloning ! 3

Let’s do Some Cloning ! 4

Applications of Limonene 5

Limonene as a Biofuel Jet A (A-1) Standards: Jet A* Ethanol Dodecane Limonene Standards Flash point ( °C ) Min: 38 51.1 14 83 43 Autoignition temp ( °C ) 210 365 203 237 Freezing Point ( °C ) Max: −40 (−47) -51 -114 -10 -74 Boiling Point ( °C ) Max: 300 (340) 280 78 216 176 Density @ 15 °C (kg/L) 0.775-0.840 0.820 0.79 0.75 0.84** Heat of Comb. (MJ/kg) Min: 42.8 43.02 29.70 44.15 45.0 Energy density (MJ/L) 35.3 23.5 33.1 37.8 Notes: * British Petroleum; ** Density@20°C; 6

Enzymatic Production of Limonene Acetyl-CoA Mevalonate HMG IPP Mevalonate – 5 Phosphate Limonene GPP FPP DMAPP Amorphadiene Martin et al . Nat. Biotech. 2003 7

Production Vectors pBbA5C Acetyl-CoA Mevalonate HMG IPP Mevalonate – 5 Phosphate OVERALL Limonene GPP Acetyl-CoA FPP FPP DMAPP Amorphadiene 8

Production Vectors pBbA5C with geranyl pyrophosphate synthase insertion pBbA5C Acetyl-CoA Mevalonate HMG IPP Mevalonate – 5 Phosphate OVERALL LIMONENE! GPP Acetyl-CoA GPP FPP FPP DMAPP Amorphadiene 9

Production Vectors Acetyl-CoA Mevalonate HMG J23102 – Limonene synthase J23102-BBa_I742111 IPP J23102 Mevalonate – 5 Phosphate LIMONENE! GPP FPP DMAPP Amorphadiene 10

Production Vectors Acetyl-CoA Mevalonate HMG Amorphadiene synthase IPP Mevalonate – 5 Phosphate LIMONENE! GPP FPP DMAPP Amorphadiene 11

Summary of Production Strains pBbA5C Acetyl-Coa Mevalonate HMG + + IPP J23102 – Limonene synthase Amorphadiene synthase Mevalonate – 5 Phosphate OR J23102 LIMONENE! GPP = = FPP DMAPP Limonene Amorphadiene Amorphadiene 12

Summary of Production Strains pBbA5C with geranyl pyrophosphate synthase insertion Acetyl-Coa Mevalonate HMG + J23102 – Limonene synthase IPP Mevalonate – 5 Phosphate J23102 LIMONENE! GPP = FPP DMAPP Limonene Amorphadiene 13

Production Assay Extracted Diluted Diluted Gas chromatography 5 ml 40 ml cultures Mass spectrometry overnight IPTG induced cultures Dodecane overlay 14

Production Assay Results (X10,000,000) (X10,000) 1.10 9.50 1.00 8.50 Total Ion Count GC-MS Similarity Search Results (X10,000) 1.00 0.90 68 Target Compound 7.50 93 0.75 (Limonene Standard) 0.80 Total Ion Count 0.50 Total Ion Count 6.50 79 53 0.70 pBbA5c + pADS Limonene 121 0.25 pBbA5C-GPPS + pADS 5.50 0.60 0.00 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 105.0 110.0 115.0 120.0 Total Ion Count (X10,000) 1.00 0.50 4.50 68 Library Standard 0.75 0.40 3.50 pBbA5c + J23102-LIMS1 0.50 93 pBbA5c + J23102 0.30 53 0.25 79 2.50 121 Dodecane 0.20 0.00 90.0 100.0 110.0 115.0 120.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 95.0 105.0 1.50 0.10 0.50 5.00 6.00 7.00 8.00 9.00 10.0 11.0 12.0 13.0 7.45 7.50 7.55 Retention Time (min) 7.60 7.65 7.70 7.75 7.80 7.85 Retention Time (min) 15

Dissecting the Problem pBbA5C Acetyl-Coa Mevalonate HMG + + J23102 – Limonene synthase IPP Amorphadiene synthase Mevalonate – 5 Phosphate Where do we start? J23102 LIMONENE! GPP = = FPP DMAPP Limonene Amorphadiene Amorphadiene 16

Central Dogma Step : DNA mRNA Protein Folding Phenotype Confirmation: Sequencing J23102 well Questionable Suspect due to Nothing characterized heterologous proteins 17

Codon Usage in E. Coli 70 60 50 Percentage of Codons 40 LimS1 CO LimS1 CO-GPPS 30 Native MalE 20 10 0 0-10 11-20. 21-30 31-40 41-50 51-60 61-70 71-80 81-90 91-100 Codon Quality 18

Troubleshooting Translation Pros Method Cons Expensive Mass Multi-variable Specific Spectrometry Functionality unknown Quantifiable Sensitive Expensive Western Blot Quantifiable Functionality unknown Protein purification In vitro assay Protein functionality Tags may alter activity Enzymatic activity 19

Translational Coupling Cassette Ribosome helicase activity melts the hairpin… The stop codon releases the ribosome… When your gene is being fully translated… Liberated RBS is availible for binding… Reporter gene is translated 20

Translational Coupling Cassette If the target gene is not translated… The RBS is sequestered and the reporter gene is not translated 21

Cloning Into the TCC Is it translating? Translation? Gene of Interest Reporter Gene (RFP) 22

Translational Coupling Cassette 23 Mendez-Perez et al. Metab Eng 2012

Characterization of the Cassette  Two genes were cloned into the cassette for characterization  E0040: green fluorescent protein • Positive control GFP-TCC-RFP  Limonene synthase with an embedded stop codon • Negative control LimS1-Stop-TCC 24

96-Well Plate Reader Assay Diluted Tecan Fluorescence plate reader 5 ml 96 well plate overnight cultures 25

96-well Characterization Data GPPS-TCC-RFP TCC-RFP 12 Hours LimS1-TCC-RFP 18 Hours Strain 24 Hours COLimS1(MS)-TCC-RFP COLimS1(NS)-TCC-RFP GFP-TCC-RFP 0 50 100 150 RFP / OD600 (au) 26

Typhoon Imager Assay Dropped Incubated culture overnight Scanned in Drop plate 5 ml typhoon imager overnight cultures 27

Typhoon Imager Data 2 1 TCC-RFP GFP-TCC-RFP 3 4 5 6 LimS1-Stop-TCC LimS1-TCC 7 COLimS1-TCC COLimS1-Stop-TCC GPPS-TCC 28

Typhoon Imager Data 2 1 TCC-RFP GFP-TCC-RFP 3 4 5 6 LimS1-Stop-TCC LimS1-TCC 7 COLimS1-TCC COLimS1-Stop-TCC GPPS-TCC 28

Typhoon Imager Data 29

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

Further Production Assays pBbA5C pBbA5C with geranyl pyrophosphate synthase insertion (X10,000,000) (X10,000,000) 1.10 1.10 1.00 1.00 0.90 0.90 Total Ion Count Total Ion Count 0.80 0.80 0.70 0.70 Limonene Limonene 0.60 0.60 0.50 0.50 0.40 0.40 pBbA5c-GPPS + J23102-COLIMS1 pBbA5c + J23102-COLIMS1 0.30 0.30 pBbA5c-GPPS + J23102 pBbA5c + J23102 Dodecane 0.20 0.20 0.10 0.10 Dodecane 5.00 5.00 6.00 6.00 7.00 7.00 8.00 8.00 9.00 9.00 10.0 10.0 11.0 11.0 12.0 12.0 13.0 13.0 Retention Time (min) Retention Time (min) 31

Conclusions and Future Steps pBbA5C with geranyl pyrophosphate synthase insertion Acetyl-Coa Mevalonate HMG + J23102 – Limonene synthase IPP  In vitro assay after  Run in vitro assay after Mevalonate – 5 Phosphate purification purification  Western blot with His-Tag J23102  Western blot with His- LIMONENE! GPP antibodies Tag antibodies =  Different production techniques FPP DMAPP Limonene Amorphadiene 32

Translational Coupling Cassette A synthetic biology tool for troubleshooting the expression of heterologous proteins Target Gene Gene of Interest Reporter Gene 33

iGEM Outreach 34

iGEM Outreach James Madison Memorial High School advanced placement biology classes 35

Lab Visits and Brainstorming Sessions Facilitating the formation of their high school iGEM team 35

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