samaneh noor department of chemical engineering the university of tulsa method to assemble...
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Samaneh NoorDepartment of Chemical Engineering
The University of Tulsa
Method to Assemble Biosynthetic Pathways in Microalgae
IPEC 2010 September 2, 2010
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Outline
• Goal of Research• Introduction and Background• Assembly of Multi-Gene Biosynthetic
Pathways into Microalgae• Preliminary Results• Current Work• Summary
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Goal of Research
• Develop a Method to Genetically Engineer Microalgae
• Multiple Gene Biochemical Pathways
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Why Microalgae?Why Microalgae?
• GRAS• Low Impact• Flexible on Water Quality• High Growth Rate• Low Production Cost• High Levels of Oils
and Hydrocarbons
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Biology of AlgaeBiology of Algae
• Aquatic, Photosynthetic Organisms– Microalgae, Macroalgae
• Microalgae– Chlamydomonas reinhardtii– Single Cell, Eukaryotic
• Mitochondrial• Nuclear• Chloroplast
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Various Microalgae and
Diatoms
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6Image source: Rosenberg, J.N., Oyler, G.A., Wilkinson, L., Betenbaugh, M.J. A green light for engineered algae: redirecting metabolism to fuel a biotechnology revolution ,Current Opinion in Biotechnology, 19 (5), pp. 430-436 (2008)
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How to Improve ProductivityHow to Improve Productivity
• Selection / Screening Techniques
• Cultivation
• Genetic / Metabolic Engineering
7http://www.mvm.uni-karlsruhe.de/img/bio/rohrreaktor_neu.jpg
http://news.cnet.com/i/bto/20080620/Seambiotic_Ponds_540x354.jpg
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Genetic and Metabolic EngineeringGenetic and Metabolic Engineering
• Nucleus
• Chloroplast
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Chloroplast of C. reinhardtiiChloroplast of C. reinhardtii
– 40% Cell Volume
– Photosynthetic Apparatus
– Metabolic Pathways
– RNA and Protein Synthesizing Systems
• Prokaryotic Organisms
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Figure from Maul J.E., Lilly J.W., Cui L., et al. The Chlamydomonas reinhardtii plastid chromosome:islands of genes in a sea of repeats. Plant Cell 14(11):2659–79 (2002)
C. reinhardtiiChloroplast Genome
203,395 bp
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Advantage of Chloroplast Advantage of Chloroplast
Parameters Chloroplast NucleusGenome Sequence Complete CompleteNumber of Genes
Expressed Multiple SingleGene Integration Site Specific Not SpecificGene Silencing Not Probable Probable
Versatility to Express genes from other
organisms
High Low
Recombination Machinery
Homologous Non Homologous
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Gene Gene Product
aadA Aminoglycoside adenyl transferaseuidA β-glucoronidaserecA RecA proteinRluc/luxCt LuciferaseaphA6 Aminoglycoside phosphotransferasegfp/GFPct Green fluorescent proteingds Geranylgeranyl pyrophosphate synthaseHSV8-lsc Single-chain antibodyFMDV.VP1 Viral proteinapc AllophycocyaninnifH Nitrogenase subunitm-saa Bovine serum amyloidlacI Lac repressor proteinhMT-2 Metallothionein-2Trail TRAIL proteinamy α-amylaseCSFV E2 E2 proteinhGAD65 Glutamic acid decarboxylase83K7C Monoclonal antibody
reporter protein
pharmaceutical protein
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Goal of Research
• Genetically Engineer Microalgae
• Multiple Gene Biochemical Pathways
– Short Term Goal» Sugar Utilization» Hydrocarbon Production
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1. Amplify Cassettes and Gene Assembly in Yeast
1 WEEK
Chloroplast DNA
Yeast ori
E. coli ori Yeast Selection
Chloroplast DNA
2. Transformation
3. Selecting Primary Transformants and Homoplasmic lines
5’ UTR Gene 3’ UTR
OE-PCR
Expression Cassette
2WEEKS
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Plasmid Assembly and Integration
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gene5’UTR 3’UTR
Vector
Chloroplast DNA
Yeast ori
E. coli ori Yeast Selection
Chloroplast DNA
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Chloroplast Transformation
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Transformed AlgaeBiolostic Particle Gun
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Homoplasmic Lines
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TAP+Kan Plates
Grow Homoplasmic Colony
80 Chloroplasts Contain Gene
1 2 43 5 6
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Gene Verification
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1=Mass Marker2=cc125-pTJ322-aphA6; aphA6 gene
3= cc125-pTJ322-aphA6-aadA; aphA6 gene4=cc125-pTJ322-aphA6-aadA; aadA gene
5=Mass Marker
120 V, 0.7% agarose, 30 min
1 3 4 52
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Growth on AntibioticsaphA6
aadA+aphA6
• One Gene Successfully and Functionally Expressed
• Two Genes Successfully and Functionally Expressed
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Growth Curvecc125-pTJ322-aphA6
Wild Type Strain
Engineered Strain
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Current Work
• Other Multiple Gene Pathway Constructions
• aphA6-phbC-phbA-phbB• aphA6-Arabinose
• Introduce Assembled Genes into Algae
• Assay for Enzyme Activity
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Summary
• Algae - a Promising Source for Fuel and Therapeutic Proteins
• Success Assembling Up to 3 Gene Pathways
• Success Integrating 3 Gene Pathways into Chloroplast Genome of Chlamydomonas reinhardtii
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Acknowledgments
Professor Tyler Johannes Azadeh M Pourmir TU Chemical Engineering Department
Maddie Laizure
Kelby Aten
Chris Dean
Kayla Kutter
Anne Campbell
22For more information please visit: http://johannes.wik.is/