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Samaneh Noor Department of Chemical Engineering The University of Tulsa

Method to Assemble Biosynthetic Pathways in Microalgae. Samaneh Noor Department of Chemical Engineering The University of Tulsa. IPEC 2010. September 2, 2010. Outline. Goal of Research Introduction and Background Assembly of Multi-Gene Biosynthetic Pathways into Microalgae

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Samaneh Noor Department of Chemical Engineering The University of Tulsa

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  1. Method to Assemble Biosynthetic Pathways in Microalgae Samaneh Noor Department of Chemical Engineering The University of Tulsa IPEC 2010 September 2, 2010

  2. Outline • Goal of Research • Introduction and Background • Assembly of Multi-Gene Biosynthetic Pathways into Microalgae • Preliminary Results • Current Work • Summary

  3. Goal of Research • Develop a Method to Genetically Engineer Microalgae • Multiple Gene Biochemical Pathways

  4. WhyMicroalgae? • GRAS • Low Impact • Flexible on Water Quality • High Growth Rate • Low Production Cost • High Levels of Oils and Hydrocarbons

  5. Biology of Algae • Aquatic, Photosynthetic Organisms • Microalgae, Macroalgae • Microalgae • Chlamydomonas reinhardtii • Single Cell, Eukaryotic • Mitochondrial • Nuclear • Chloroplast Various Microalgae and Diatoms

  6. Products from Microalgae Image 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)

  7. How to Improve Productivity • Selection / Screening Techniques • Cultivation • Genetic / Metabolic Engineering http://news.cnet.com/i/bto/20080620/Seambiotic_Ponds_540x354.jpg http://www.mvm.uni-karlsruhe.de/img/bio/rohrreaktor_neu.jpg

  8. Genetic and Metabolic Engineering • Nucleus • Chloroplast

  9. Chloroplast of C. reinhardtii • 40% Cell Volume • Photosynthetic Apparatus • Metabolic Pathways • RNA and Protein Synthesizing Systems • Prokaryotic Organisms C. reinhardtii Chloroplast Genome 203,395 bp 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)

  10. Advantage of Chloroplast

  11. Genes Expressed in C. reinhardtii Chloroplast reporter protein pharmaceutical protein

  12. Goal of Research • Genetically Engineer Microalgae • Multiple Gene Biochemical Pathways • Short Term Goal • Sugar Utilization • Hydrocarbon Production

  13. 1. Amplify Cassettes and Gene Assembly in Yeast 5’ UTR Gene 3’ UTR 1 W E E K OE-PCR Expression Cassette 2 W E E K S 2. Transformation 3. Selecting Primary Transformants and Homoplasmic lines Yeast ori E. coli ori Yeast Selection Chloroplast DNA Chloroplast DNA

  14. Plasmid Assembly and Integration 5’UTR gene 3’UTR Vector Yeast ori E. coli ori Yeast Selection Chloroplast DNA Chloroplast DNA

  15. Chloroplast Transformation Transformed Algae Biolostic Particle Gun

  16. TAP+Kan Plates Homoplasmic Lines 1 2 3 4 6 5 Grow Homoplasmic Colony 80 Chloroplasts Contain Gene

  17. Gene Verification 1 2 3 4 5 120 V, 0.7% agarose, 30 min 1=Mass Marker 2=cc125-pTJ322-aphA6; aphA6 gene 3= cc125-pTJ322-aphA6-aadA; aphA6 gene 4=cc125-pTJ322-aphA6-aadA; aadA gene 5=Mass Marker

  18. Growth on Antibiotics aphA6 • One Gene Successfully and • Functionally Expressed aadA+aphA6 • Two Genes Successfully and • Functionally Expressed

  19. Growth Curvecc125-pTJ322-aphA6 Engineered Strain Wild Type Strain

  20. Current Work • Other Multiple Gene Pathway Constructions • aphA6-phbC-phbA-phbB • aphA6-Arabinose • Introduce Assembled Genes into Algae • Assay for Enzyme Activity

  21. 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

  22. Acknowledgments • Professor Tyler Johannes • Azadeh M Pourmir • TU Chemical Engineering Department • Maddie Laizure • Kelby Aten • Chris Dean • Kayla Kutter • Anne Campbell For more information please visit: http://johannes.wik.is/

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