Abstract

Danielle Bartz, Yishen Cai, Charles Johnson, Chris Johnson, Ryan Leenay, Klaus Lovendahl, Menuka Samaranayake, Eric Walters, Chelsea Webster, Michael Zaiken

Advisors: Brian Pfleger, Ph.D, Matthew Copeland, Ph.D

In synthetic biology, a powerful method for the production of novel metabolites is the expression of heterologous genes in Escherichia coli. A common challenge when using non-native genes in metabolic engineering is determining if they are being properly expressed. To address this issue, we have constructed a BioFusion compatible system for testing the translation of a gene of interest1. This system couples the translation of the target gene to a fluorescent reporter gene. Fluorescence will only be detected when the target gene is entirely translated. This construct enables synthetic biologists to quickly determine if a gene is being expressed without the need for costly antibodies or analytical instruments (e.g. mass spectrometry). Currently, we are utilizing this cassette to troubleshoot the expression of limonene synthase, an enzyme that catalyzes the production of limonene, a monoterpene with potential as a renewable jet fuel.

The Solution: Translational Coupling Cassette

What are we trying to make? Enzymatic Production of Limonene

Results

Conclusions

Motivation

Translational Coupling Cassette:

A tool for evaluating the translation of heterologous proteins in Escherichia coli

Limonene Production Assay

2Martin

96-well plate fluorescence data (Tecan Plate Reader)

GFP

E X

S P

Digestion Ligation

Transformation

A. Target gene translated RFP translated/ generated

B. Target gene not translated hairpin prevents RFP translation/ generation

Acetyl-CoA

Limonene (Not Detected) GPP

IPP Mevalonate – 5 Phosphate

Mevalonate HMG

Amorphadiene (Detected)

DMAPP

FPP

Hairpin Design

A. When target gene is translated, the hairpin is melted, allowing translation of the RFP reporter gene, and generation

of RFP fluorescence.

B. If translation of target gene is unsuccessful, the intact hairpin prevents translation of the RFP

reporter gene.

Typhoon image (Typhoon Imager)

1Mendez-Perez

Special thanks to the UW-Biochemistry department for use of Typhoon Imager

Limonene Production Assay GC-MS Results

pBbA5c + pADS �pBbA5C-GPPS + pADS �

Retention Time (min) �

Tota

l Ion

Cou

nt�

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-GPPS + J23102-COLIMS1 �pBbA5c-GPPS + J23102 �

Tota

l Ion

Cou

nt�

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-LIMS1 �pBbA5c + J23102 �

Retention Time (min) �

Tota

l Ion

Cou

nt�

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) �

pBbA5c + J23102-COLIMS1 �pBbA5c + J23102 �

Retention Time (min) �

Tota

l Ion

Cou

nt�

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) �

(X10,000) �

(X10,000) �

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 �

Tota

l Ion

Cou

nt�

Tota

l Ion

Cou

nt�

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

TCC •  Cassette is functioning as designed

•  LimS1 is not being translated •  COLimS1 and GPPS are being translated

Level   DNA   RNA

  Protein ? Phenotype

Confirmation Sequence confirmed

Unlikely; reliable promoter Confirmed via TCC

No limonene detected

Status of Limonene Production

Future steps •  In vitro assay of GPPS and COLimS1 •  Western Blot for His-tagged proteins

Pathway Confirmation Mevalonate pathway functions to ispA

(amorphadiene synthesized)

1Mendez-Perez et. al. 2012 Metabolic Engineering 2Martin et. al. 2003 Nature Biotechnology 3Lücker et. al, 2002. European Journal of Biochemistry

References

? Imagine you have a gene that you want to insert

into a plasmid

And the transformation is confirmed by sequencing and

restriction digests

But you are still not achieving your desired phenotype.

How can you troubleshoot this?

Limonene Production Analysis   DNA   RNA ? Protein ? Phenotype

Sequence confirmed Unlikely; reliable promoter Translated? No limonene detected

GFP-TCC-RFP

COLimS1-NS-TCC COLimS1-MS-TCC*

LimS1-NS-TCC LimS1-Stop-TCC*

TCC-RFP

GPPS-TCC

•  TCC positive control (GFP-TCC-RFP) works as designed

•  TCC for codon optimized limonene synthase (COLimS1-TCC) exhibits RFP fluorescence

•  TCC for geranyl pyrophosphate synthase (GPPS-TCC) exhibits RFP fluorescence

•  Base TCC-RFP (Part: BBa_K762000) is available for use by all iGEM teams

0 20 40 60 80 100 120 140 160

GFP-TCC-RFP

COLimS1(NS)-TCC-RFP

COLimS1(MS)-TCC-RFP

LimS1-TCC-RFP

TCC-RFP

GPPS-TCC-RFP

RFP / OD600 (au)

Stra

ins

12 Hours

18 Hours

24 Hours

Typhoon image analysis (Pixel density analysis using Photoshop)

1 2

3 4

5 6

7 7

1 2

3 4

5 6

*Negative Controls: Stop indicates presence of stop codon in target gene Drop plate

(Camera image)