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 interest 1 . 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. Translational Coupling Cassette 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 2 Martin RFP Fluorescence Strains 96-Well plate fluorescence data (Tecan Plate Reader) Typhoon image analysis (Pixel density analysis by Photoshop) Drop plate (Nikon camera image) A. Target gene translated RFP translated/ generated B. Target gene not translated hairpin prevents RFP translation/ generation ? Acetyl-CoA Limonene GPP IPP Mevalonate – 5 Phosphate Mevalonate HMG Amorphadiene 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. GFP-TCC-RFP COLimS1-TCC COLimS1-Stop-TCC* LimS1-TCC LimS1-Stop-TCC* TCC-RFP Typhoon image (Typhoon Imager) • TCC positive control (GFP-TCC-RFP) fluoresces as designed • TCC for codon optimized limonene synthase (COLimS1-TCC) exhibits RFP fluorescence • Base TCC-RFP (Part: BBa_K762000) is available for use by all iGEM teams 1 Mendez-Perez Special thanks to the UW-Biochemistry department for use of Typhoon Imager *Negative Controls: Stop indicates presence of stop codon in target gene Limonene Production Assay GC-MS Results 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-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-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) 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) (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 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 TCC • Cassette is functioning as designed • LimS1 is not being translated • COLims1 is being translated Level DNA RNA Protein ? Phenotype Confirmation Sequence confirmed Unlikely; reliable promoter Confirmed via TCC No limonene detected Limonene Production Troubleshooting Methods Future steps • In vitro assay of GPPS and COLimS1 • Clone GPPS into TCC • Western Blot for His-tagged proteins Pathway Confirmation Mevalonate pathway functions to ispA (amorphadiene synthesized) 1 Mendez-Perez et. al. 2012 Metabolic Engineering 2 Martin et. al. 2003 Nature Biotechnology 3 Lücker et. al, 2002. European Journal of Biochemistry References 0 25 50 75 100 125 150 GFP-TCC-RFP COLimS1(NS) COLimS1(MS) TCC-RFP LimS1(NS) RFP / OD 600 (au) Strain 12 Hours 18 Hours 24 Hours
Welcome message from author
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
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.
Translational Coupling Cassette
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
RFP Fluorescence
Stra
ins
96-Well plate fluorescence data (Tecan Plate Reader)
Typhoon image analysis (Pixel density analysis by Photoshop)
Drop plate (Nikon camera image)
Digestion Ligation
Transformation
A. Target gene translated RFP translated/ generated
B. Target gene not translated hairpin prevents RFP translation/ generation
?
Acetyl-CoA
Limonene GPP
IPP Mevalonate – 5 Phosphate
Mevalonate HMG
Amorphadiene 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.
GFP-TCC-RFP
COLimS1-TCC COLimS1-Stop-TCC*
LimS1-TCC LimS1-Stop-TCC*
TCC-RFP
Typhoon image (Typhoon Imager)
• TCC positive control (GFP-TCC-RFP) fluoresces as designed
• TCC for codon optimized limonene synthase (COLimS1-TCC) exhibits RFP fluorescence
• Base TCC-RFP (Part: BBa_K762000) is available for use by all iGEM teams
1Mendez-Perez
Special thanks to the UW-Biochemistry department for use of Typhoon Imager
*Negative Controls: Stop indicates presence of stop codon in target gene
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 is being translated
Level ü DNA ü RNA ü Protein ? Phenotype
Confirmation Sequence confirmed Unlikely; reliable promoter Confirmed via TCC No limonene detected
Limonene Production Troubleshooting Methods
Future steps • In vitro assay of GPPS and COLimS1
• Clone GPPS into TCC • 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 0 25 50 75 100 125 150
GFP-TCC-RFP
COLimS1(NS)
COLimS1(MS)
TCC-RFP
LimS1(NS)
RFP / OD600 (au)
Stra
in
12 Hours
18 Hours
24 Hours
Related Documents
