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!

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Let’s do Some Cloning!

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Let’s do Some Cloning!

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Let’s do Some Cloning!

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Applications of Limonene

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Limonene as a Biofuel

Standards: Jet A (A-1) Standards Jet A* Ethanol Dodecane Limonene 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;

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Enzymatic Production of Limonene

Amorphadiene DMAPP Acetyl-CoA Limonene GPP IPP Mevalonate – 5 Phosphate Mevalonate HMG FPP

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Martin et al. Nat. Biotech. 2003

Production Vectors

Acetyl-CoA Amorphadiene DMAPP Limonene GPP IPP Mevalonate – 5 Phosphate Mevalonate HMG FPP pBbA5C Acetyl-CoA FPP

OVERALL

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

OVERALL

GPP

Production Vectors

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Acetyl-CoA Amorphadiene DMAPP LIMONENE! GPP IPP Mevalonate – 5 Phosphate Mevalonate HMG FPP

J23102

J23102 – Limonene synthase J23102-BBa_I742111

Production Vectors

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

Production Vectors

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Summary of Production Strains

Acetyl-Coa Amorphadiene DMAPP LIMONENE! GPP IPP Mevalonate – 5 Phosphate Mevalonate HMG FPP pBbA5C

+

Amorphadiene synthase

=

Amorphadiene

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+ =

J23102

J23102 – Limonene synthase Limonene OR

Acetyl-Coa Amorphadiene DMAPP LIMONENE! GPP IPP Mevalonate – 5 Phosphate Mevalonate HMG FPP

+ =

pBbA5C with geranyl pyrophosphate synthase insertion

J23102

J23102 – Limonene synthase Limonene

Summary of Production Strains

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Production Assay

5 ml

• vernight

cultures Diluted 40 ml cultures IPTG induced Dodecane overlay Extracted Diluted Gas chromatography Mass spectrometry

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Production Assay Results

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Library Standard Target Compound

(Limonene Standard) 0.00 0.50 1.00 0.75 0.25 0.00 0.50 1.00 0.75 0.25

Total Ion Count Total Ion Count

GC-MS Similarity Search Results

70.0 90.0 80.0 60.0 55.0 65.0 75.0 85.0 95.0 100.0 120.0 115.0 110.0 105.0 70.0 90.0 80.0 60.0 55.0 65.0 75.0 85.0 95.0 100.0 120.0 115.0 110.0 105.0

(X10,000) (X10,000)

53 68 79 93 121 53 68 79 93 121

pBbA5c + pADS pBbA5C-GPPS + pADS Retention Time (min) Total Ion Count

4.50 2.50 6.50 8.50 9.50 7.50 5.50 3.50 1.50 0.50 7.60 7.80 7.70 7.50 7.45 7.55 7.65 7.75 7.85

(X10,000)

pBbA5c + J23102-LIMS1 pBbA5c + J23102 Retention Time (min) Total Ion Count

Limonene Dodecane

8.00 12.0 10.0 6.00 5.00 7.00 9.00 11.0 13.0 0.60 0.20 0.40 0.80 1.00 1.10 0.90 0.70 0.50 0.30 0.10

(X10,000,000)

Dissecting the Problem

Acetyl-Coa Amorphadiene DMAPP LIMONENE! GPP IPP Mevalonate – 5 Phosphate Mevalonate HMG FPP pBbA5C

+

Amorphadiene synthase

=

Amorphadiene

+ =

J23102

J23102 – Limonene synthase Limonene

Where do we start?

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Central Dogma

Confirmation: Sequencing J23102 well Questionable Suspect due to Nothing

characterized heterologous proteins

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Step:

DNA mRNA Protein Folding Phenotype

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

Codon Usage in E. Coli

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Troubleshooting Translation

Cons

Expensive Multi-variable Functionality unknown Expensive Functionality unknown Protein purification Tags may alter activity

Method

Mass Spectrometry Western Blot In vitro assay

Pros

Specific Quantifiable Sensitive Quantifiable Protein functionality Enzymatic activity

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Translational Coupling Cassette

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

Translational Coupling Cassette

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If the target gene is not translated…

The RBS is sequestered and the reporter gene is not translated

Cloning Into the TCC

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Gene of Interest Reporter Gene (RFP) Is it translating? Translation?

Translational Coupling Cassette

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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
• Limonene synthase with an embedded stop codon
• Negative control

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GFP-TCC-RFP LimS1-Stop-TCC

96-Well Plate Reader Assay

5 ml

• vernight

cultures 96 well plate Tecan Fluorescence plate reader

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Diluted

96-well Characterization Data

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50 100 150

GFP-TCC-RFP COLimS1(NS)-TCC-RFP COLimS1(MS)-TCC-RFP LimS1-TCC-RFP TCC-RFP GPPS-TCC-RFP

RFP / OD600 (au) Strain 12 Hours 18 Hours 24 Hours

Typhoon Imager Assay

5 ml

• vernight

cultures Dropped culture Drop plate Scanned in typhoon imager Incubated

• vernight

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1 2 3 4 5 6 7

Typhoon Imager Data

GFP-TCC-RFP COLimS1-TCC LimS1-TCC LimS1-Stop-TCC TCC-RFP COLimS1-Stop-TCC GPPS-TCC

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7 1 2 3 4 5 6

Typhoon Imager Data

GFP-TCC-RFP COLimS1-TCC LimS1-TCC LimS1-Stop-TCC TCC-RFP COLimS1-Stop-TCC GPPS-TCC

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Typhoon Imager Data

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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)

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Further Production Assays

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pBbA5c-GPPS + J23102-COLIMS1 pBbA5c-GPPS + J23102 Total Ion Count Retention Time (min)

Limonene Dodecane

0.60 0.20 0.40 0.80 1.00 1.10 0.90 0.70 0.50 0.30 0.10 8.00 12.0 10.0 6.00 5.00 7.00 9.00 11.0 13.0

(X10,000,000)

pBbA5c + J23102-COLIMS1 pBbA5c + J23102 Retention Time (min) Total Ion Count

Limonene Dodecane

0.60 0.20 0.40 0.80 1.00 1.10 0.90 0.70 0.50 0.30 0.10 8.00 12.0 10.0 6.00 5.00 7.00 9.00 11.0 13.0

(X10,000,000)

pBbA5C with geranyl pyrophosphate synthase insertion pBbA5C

Conclusions and Future Steps

Acetyl-Coa Amorphadiene DMAPP LIMONENE! GPP IPP Mevalonate – 5 Phosphate Mevalonate HMG FPP

+ =

pBbA5C with geranyl pyrophosphate synthase insertion

J23102

J23102 – Limonene synthase Limonene

• Run in vitro assay after

purification

• Western blot with His-

Tag antibodies

• In vitro assay after

purification

• Western blot with His-Tag

antibodies

• Different production

techniques

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Gene of Interest

Translational Coupling Cassette

A synthetic biology tool for troubleshooting the expression of heterologous proteins

Reporter Gene Target Gene

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iGEM Outreach

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iGEM Outreach

James Madison Memorial High School advanced placement biology classes

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Lab Visits and Brainstorming Sessions

Facilitating the formation of their high school iGEM team

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Acknowledgements

Special thank you to: Brian Pfleger PhD Matthew Copeland PhD Jennifer Reed PhD Sophia Weerth UW-Madison Biochemistry Dept. UW-Madison Engineering Dept.

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