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
Presentation for EnergieCampus 2016
07.11.2016
Botanical Institute
Faculty of Chemistry and Biosciences
3
Background
Fabian Nies
Presentation for EnergieCampus 2016
07.11.2016
Botanical Institute
Faculty of Chemistry and Biosciences
4
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
Presentation for EnergieCampus 2016
07.11.2016
Botanical Institute
Faculty of Chemistry and Biosciences
5
Background
Fabian Nies
Presentation for EnergieCampus 2016
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
Faculty of Chemistry and Biosciences
6
Objective of research
Phormidium
Cyanobacteria
Order: Oscillatoriales
Filamentous growth in adherent
and in suspension cell culture
Motile
Fast growing
Robust
Growing under strong light
conditions
Fabian Nies
Presentation for EnergieCampus 2016
07.11.2016
Botanical Institute
Faculty of Chemistry and Biosciences
7
Objective of research
Ethanol synthesis
Fabian Nies
Presentation for EnergieCampus 2016
07.11.2016
Botanical Institute
Faculty of Chemistry and Biosciences
8
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
Presentation for EnergieCampus 2016
07.11.2016
Botanical Institute
Faculty of Chemistry and Biosciences
9
RESULTS Transformation of Phormidium
Fabian Nies
Presentation for EnergieCampus 2016
07.11.2016
Botanical Institute
Faculty of Chemistry and Biosciences
10
Transformation of Phormidium
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
Presentation for EnergieCampus 2016
07.11.2016
Botanical Institute
Faculty of Chemistry and Biosciences
11
Transformation of Phormidium
Insertion of the KanR gene into the homologous sequences of the
strain HE10JO
Fabian Nies
Presentation for EnergieCampus 2016
07.11.2016
Botanical Institute
Faculty of Chemistry and Biosciences
12
Transformation of Phormidium
Establishment of the transformation method (via electroporation)
Mix cells and DNA
Electroshock
Regeneration
Selection
Validation
Fabian Nies
Presentation for EnergieCampus 2016
07.11.2016
negative control pGEM-2-KanR pGEM-55-KanR pGEM-7-KanR
Botanical Institute
Faculty of Chemistry and Biosciences
13
Transformation of Phormidium
Establishment of the transformation method (via electroporation)
Fabian Nies
Presentation for EnergieCampus 2016
07.11.2016
negative control pGEM-2-KanR pGEM-55-KanR pGEM-7-KanR
0 70 120
Kanamycin [µg/ml]
Botanical Institute
Faculty of Chemistry and Biosciences
14
Transformation of Phormidium
Transformation method (electroporation)
Fabian Nies
Presentation for EnergieCampus 2016
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
Faculty of Chemistry and Biosciences
15
Transformation of Phormidium
Validation of the Transformation
Fabian Nies
Presentation for EnergieCampus 2016
07.11.2016
Botanical Institute
Faculty of Chemistry and Biosciences
16
Transformation of Phormidium
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
Presentation for EnergieCampus 2016
07.11.2016
Botanical Institute
Faculty of Chemistry and Biosciences
17
Perspectivs
Bioreactor
Fabian Nies
Presentation for EnergieCampus 2016
07.11.2016
Botanical Institute
Faculty of Chemistry and Biosciences
18
Perspectivs
Bioreactor
Fabian Nies
Presentation for EnergieCampus 2016
07.11.2016
sunlight
medium
Phormidium
Botanical Institute
Faculty of Chemistry and Biosciences
19
Summary
Fabian Nies
Presentation for EnergieCampus 2016
07.11.2016
Phormidium
Transformation method
Outlook: Characterization of promoters
Long term goal: Optimized production of ethanol in bioreactors
Botanical Institute
Faculty of Chemistry and Biosciences
20
Fabian Nies
Presentation for EnergieCampus 2016
07.11.2016