recombinant synthesis of organic compounds in cyanobacteria€¦ · fabian nies presentation for...

KIT – The Research University in the Helmholtz Association

Botanical Institute, Faculty of Chemistry and Biosciences

www.kit.edu

Recombinant synthesis of organic compounds in cyanobacteria

Presentation for EnergieCampus 2016

Botanical Institute

Faculty of Chemistry and Biosciences

2

Structure

Background

Biofuels and microalgae

Objective of research

Results

Transformation of Phormidium

Outlook

Summary

Fabian Nies


07.11.2016

Botanical Institute


3

Background

Fabian Nies


07.11.2016

Botanical Institute


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Background

Biofuels

First generation

Fuels extracted from the fruit of agriculture corps

Second generation

Fuels extracted from the whole plant

Third generation

Generation of fuels with microalgae

Fabian Nies


07.11.2016

Botanical Institute


5

Background

Fabian Nies


07.11.2016

• Higher growth rates

• Whole organism is photosynthetic active

• Cultivation is independent from soil quality

• Less productions steps from the organism to the product

• CO2 can be recycled

• Cultivation in closed systems is unproblematic concerning GMOs

Advantages of microalgaes over rooted

plants

Botanical Institute


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Phormidium

Cyanobacteria

Order: Oscillatoriales

Filamentous growth in adherent

and in suspension cell culture

Motile

Fast growing

Robust

Growing under strong light

conditions

Fabian Nies


07.11.2016

Botanical Institute


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Ethanol synthesis

Fabian Nies


07.11.2016

Botanical Institute


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Aim of this work

Effective, transgenic production of ethanol and possibly other

compounds in Phormidium

Transformation method for Phormidium via integration into the genome

Production of ethanol in Phormidium

Optimized ethanol production on the level of genetic design and cultivation

Fabian Nies


07.11.2016

Botanical Institute


9

RESULTS Transformation of Phormidium

Fabian Nies


07.11.2016

Botanical Institute


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Insertion of the KanR gene into the homologous sequences of the

strain HE10JO

Cloning the homologous sequences into a plasmid

Insertion of the KanR gene into the homologous sequences on the vector

Fabian Nies


07.11.2016

Botanical Institute


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Insertion of the KanR gene into the homologous sequences of the

strain HE10JO

Fabian Nies


07.11.2016

Botanical Institute


12


Establishment of the transformation method (via electroporation)

Mix cells and DNA

Electroshock

Regeneration

Selection

Validation

Fabian Nies


07.11.2016

negative control pGEM-2-KanR pGEM-55-KanR pGEM-7-KanR

Botanical Institute


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Establishment of the transformation method (via electroporation)

Fabian Nies


07.11.2016

negative control pGEM-2-KanR pGEM-55-KanR pGEM-7-KanR

0 70 120

Kanamycin [µg/ml]

Botanical Institute


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Transformation method (electroporation)

Fabian Nies


07.11.2016

negative control pGEM-2-KanR pGEM-55-KanR pGEM-7-KanR Kanamycin 0 µg/ml 20000 µg/ml

Transformed

cells

Wild type

Botanical Institute


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Validation of the Transformation

Fabian Nies


07.11.2016

Botanical Institute


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Subsequent experiments and perspectives

Facilitate efficient gene expression in Phormidium

GFP assays and PDC and ADH cloning with promising promoters

Advancement of the transformation protocol

Testing other Phormidium strains

Optimization and variations

Optimization on the genetic level and in bioreactors

Fabian Nies


07.11.2016

Botanical Institute


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Perspectivs

Bioreactor

Fabian Nies


07.11.2016

Botanical Institute


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Perspectivs

Bioreactor

Fabian Nies


07.11.2016

sunlight

medium

Phormidium

Botanical Institute


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Summary

Fabian Nies


07.11.2016

Phormidium

Transformation method

Outlook: Characterization of promoters

Long term goal: Optimized production of ethanol in bioreactors

Botanical Institute


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Fabian Nies


07.11.2016

recombinant synthesis of organic compounds in cyanobacteria€¦ · fabian nies presentation for...

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