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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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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

  • Preliminary Results

  • Current Work

  • Summary


Goal of Research

  • Develop a Method to Genetically Engineer Microalgae

    • Multiple Gene Biochemical Pathways


WhyMicroalgae?

  • GRAS

  • Low Impact

  • Flexible on Water Quality

  • High Growth Rate

  • Low Production Cost

  • High Levels of Oils

    and Hydrocarbons


Biology of Algae

  • Aquatic, Photosynthetic Organisms

    • Microalgae, Macroalgae

  • Microalgae

    • Chlamydomonas reinhardtii

    • Single Cell, Eukaryotic

      • Mitochondrial

      • Nuclear

      • Chloroplast

Various Microalgae and Diatoms


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)


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


Genetic and Metabolic Engineering

  • Nucleus

  • Chloroplast


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)


Advantage of Chloroplast


Genes Expressed in C. reinhardtii Chloroplast

reporter protein

pharmaceutical protein


Goal of Research

  • Genetically Engineer Microalgae

    • Multiple Gene Biochemical Pathways

      • Short Term Goal

        • Sugar Utilization

        • Hydrocarbon Production


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


Plasmid Assembly and Integration

5’UTR

gene

3’UTR

Vector

Yeast ori

E. coli ori

Yeast Selection

Chloroplast DNA

Chloroplast DNA


Chloroplast Transformation

Transformed Algae

Biolostic Particle Gun


TAP+Kan Plates

Homoplasmic Lines

1

2

3

4

6

5

Grow Homoplasmic Colony

80 Chloroplasts Contain Gene


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


Growth on Antibiotics

aphA6

  • One Gene Successfully and

  • Functionally Expressed

aadA+aphA6

  • Two Genes Successfully and

  • Functionally Expressed


Growth Curvecc125-pTJ322-aphA6

Engineered Strain

Wild Type Strain


Current Work

  • Other Multiple Gene Pathway Constructions

    • aphA6-phbC-phbA-phbB

    • aphA6-Arabinose

  • Introduce Assembled Genes into Algae

  • Assay for Enzyme Activity


  • 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


    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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