Analysis of Peroxisomal Lipid Metabolism in
the Oleaginous Microalga Nannochloropsis
and Development of Synthetic Biolog...
Contents
• Introduction
• Thesis goal
• Methods
• Result
• Summary
• Future work
Introduction
Algae
• Algae are simple plants that can range from the
microscopic (microalgae), to large seaweeds
(macr...
Introduction
Introduction
Why algae
• Grow fast
• High biofuel yields
• Consume CO2
• Do not compete with agriculture
• Microalga...
Introduction
Nannochloropsis sp.
• Unicellular
• Autotrophic
• Most species live in salt water
• High growth rate
• ...
Introduction
Why Nannochloropsis gaditana?
Biomass composition of N. gaditana (Source: Radakovits
et al., 2012)
Compar...
Introduction
Development of Algal Industry
• Modern-day algal research and industry focus
production of high value alga...
Introduction
Algal lipids
• According to Fachy et. al. , “Lipids are hydrophobic
or amphipathic small molecules that ma...
Introduction
Peroxisomes
Small (0.5-1 μm), single membrane-bound organelles
• Functions:
– Fatty acid β-oxidation
– R...
Introduction
Synthetic Biology
• Synthetic biology is,
– the design and construction of new biological
parts and syste...
Introduction
Predicted PTS1 proteins
Int.
#
Annotation Predicted
PTS1
Acronym Length
P2 arogenate dehydrogenase PKL...
Thesis goals
1. Cloning of Full-lengths CDS or C-terminal exons of predicted
PTS1 Proteins in N. gaditana.
2. Subcellul...
Methods
Approaches used
• Isolation of Genomic DNA from Nannochloropsis
gaditana
• Cloning of 4 PTS1 predicted genes f...
Methods
Cloning of N. gaditana genes predicted to
have PTS1
• Cloning of CDS into pJET1.2 cloning vector
– 3 Full-leng...
Methods
Subcellular localization of predicted PTS1 Proteins
1. Coating gold particles with DNA
– 40 ng/μl plasmid DNA
...
Methods
Genomic transformation of Nannochloropsis
• By electroporation
• Using pSelect100 vector
– Linearized by BamHI...
Methods
Cloning of inducible Nannochloropsis
promoters
• Modification of pSELECT100 vector
• Making an expression vect...
Results
PCR amplification of Nannochloropsis
gaditana genes
• Ng_AroDH: 2355bp
• Ng_GOX: 750 bp
• Ng_ EAP: 1458 bp
•...
Results
2.2.Vectors
2.2.1.pCAT
The pCAT is a plant expression vector. Which localization experiments in the Reumann lab...
Results
PCR and restriction digestion analysis
of Plasmid
Plasmid analysis of inserts in pJET vector
• Ng_AroDH: 2355b...
• Positive plasmids were sent for sequencing.
• Sequencing result analyzed by blast and multiple
alingments
• Resulting...
Results
Sub-cloning of CDS/C-terminal exons in
the back of EYFP in EYFP/pCAT vector
• Restriction digestion of insert a...
Results
E coli colonies transformed with EYFP-Ng_EAP/pCAT on LB
ampicillin plates
Digestion and PCR analysis of plasmid...
Subcellular localization studies
by transient expression in onion
cells
Experimental validation
of predicted PTS1 by i...
Results
Subcellular localization of selected
Nannochloropsis gaditana proteins
carrying predicted PTS1 tripeptides
Int...
Result
Genomic transformation of N. oceanica CCMP1779
27
N. Oceanica
½ salinity f/2 agar plates
containing 50 μg/ml
...
Results
Subcloning of inducible Nannochloropsis
promoters
• The pSELECT plasmid obtained from Prof. Dr. C.
Benning con...
Results
Subcloning of inducible Nannochloropsis
promoters
• Amplification of lipid droplet
surface proteins from
pSEL...
Results
Subcloning of inducible Nannochloropsis promoters
E. coli transformed with
PRO_Ngad_LDSP/pJET (B) and
PRO_Noce...
Subcloning of inducible
Nannochloropsis promoters
E. coli transformed with Plasmid analysis of PRO_Noce_LDSP/pSELECT10
...
Manual analysis searching was done for the KpnI site of the forward
subcloning primer (GTAGGTACC GAGGTCC TGTTTGGATT TTTTG...
Result
Sequencing analysis of
PRO_Noce_LDSP/pSELECT100
Electropherogram read using Finch TV program
Summary
• 3 predicted PTS1 proteins of interest from
Nannochloropsis gaditana were sub-cloned into pJET1.2:
Ng_GOX, Ng_...
Future work
1. Sub clone N. gaditana LDSP Promoter from pJET1.2 into
pSELECT
2. Reproduction of nuclear transformation ...
Acknowledgements
• Prof. S. Reumann
• Manish Budathoki
• Dmitry Kechasov
• Dr. G. Chowdhary
• Eli Drange Vee
• All l...
of 36

Nannochloropsis gaditana, Master thesis

Analysis of Peroxisomal Lipid Metabolism in the Oleaginous Microalga Nannochloropsis and Development of Synthetic Biology Tools for Genetic Engineering
Published on: Mar 3, 2016
Published in: Science      
Source: www.slideshare.net


Transcripts - Nannochloropsis gaditana, Master thesis

  • 1. Analysis of Peroxisomal Lipid Metabolism in the Oleaginous Microalga Nannochloropsis and Development of Synthetic Biology Tools for Genetic Engineering Master’s thesis by Amit Kumar Sharma Supervisor: Prof. Sigrun Reumann Faculty of Science and Technology Department of Mathematics and Natural Sciences University of Stavanger 20. August 2014
  • 2. Contents • Introduction • Thesis goal • Methods • Result • Summary • Future work
  • 3. Introduction Algae • Algae are simple plants that can range from the microscopic (microalgae), to large seaweeds (macroalgae). • Most algae contain chlorophyll • Microalgae- cyanobacteria and green, brown and red algae • Algae –high levels of oils, carbohydrates, sugars and proteins • Base of aquatic food chain
  • 4. Introduction
  • 5. Introduction Why algae • Grow fast • High biofuel yields • Consume CO2 • Do not compete with agriculture • Microalgal biomass can be used for fuel, feed and food • Purify wastewater Crop Oil yield (gallon/acre) Corn 18 Cotton 35 Soybean 48 Mustard seed 61 Sunflower 102 Rapeseed/Canola 127 Jatropha 202 Oil palm 635 Algae 50g/m²/day at 50% triglycerides 1,200 Comparison of potential oil yields of algae. (Source: Pienkos & Darzins, 2009)
  • 6. Introduction Nannochloropsis sp. • Unicellular • Autotrophic • Most species live in salt water • High growth rate • Sequenced genomes for 2 species available • High lipid concentration (EPA + TAG) under stress conditions (Hoffmann et al., 2010) EPA: Eicosapentaenoic acid. TAG: Triacylglycerol
  • 7. Introduction Why Nannochloropsis gaditana? Biomass composition of N. gaditana (Source: Radakovits et al., 2012) Comparison of N. gaditana lipid production rates with other algae (Source: Radakovits et al., 2012)
  • 8. Introduction Development of Algal Industry • Modern-day algal research and industry focus production of high value algal products • Require huge investment, algae experts, modified oleaginous strains • Top 5 leading companies: Algenol Biofuels, Solix Biofuels, Sapphir Biofuels, Energy Solazyme. • Biofuel scientist are engineering different strains of algae at the molecular level.
  • 9. Introduction Algal lipids • According to Fachy et. al. , “Lipids are hydrophobic or amphipathic small molecules that may originate entirely or in a part by carbanion–based condensations of thioesters and/or by carbocation-based condensations of isoprene units”. • Algal lipids can be divided into two main groups: the non-polar lipids and the polar lipids.
  • 10. Introduction Peroxisomes Small (0.5-1 μm), single membrane-bound organelles • Functions: – Fatty acid β-oxidation – ROS metabolism – Photorespiration • Two major targeting signals: – PTS1: Peroxisome Targeting Signal 1 (C-terminal tripeptide) – PTS2: Peroxisome Targeting Signal 2 (N-terminal nonapeptide) Anatomy of peroxisome. UNSW Cell Biology, 2008 (http://cellbiology.med.unsw.edu.au/units/scie nce/lecture0806.htm)
  • 11. Introduction Synthetic Biology • Synthetic biology is, – the design and construction of new biological parts and systems, and – the re-design of existing, natural biological systems for useful purposes. • Algae synthetic biology are lagging behind compared to other model organisms • Tools for synthetic biology: cloning promoters and reporter genes, transformation, bioinformatics.
  • 12. Introduction Predicted PTS1 proteins Int. # Annotation Predicted PTS1 Acronym Length P2 arogenate dehydrogenase PKL> Ng_AroDH 785 aa P3 peroxisomal glycolate oxidase SKI> Ng_GOX 250 aa (from C-terminal) Full length = 390 aa P8 embryogenesis-associated protein emb8 SRL> Ng_EAP 486 aa P9 acyl- oxidase ARL> Ng_AOX 751 aa
  • 13. Thesis goals 1. Cloning of Full-lengths CDS or C-terminal exons of predicted PTS1 Proteins in N. gaditana. 2. Subcellular localization of predicted Nannochloropsis gaditana PTS1 proteins in onion epidermal cells. 3. Reproduction of nuclear transformation of Nannochloropsis oceanica using the vector pSELECT100. 4. Subcloning of the LDSP promoters from N. oceanica into pSELECT100 to create a 2nd expression cassette in pSELECT100. 5. Cloning of the LDSP promoter from N. gaditana into the pJET vector.
  • 14. Methods Approaches used • Isolation of Genomic DNA from Nannochloropsis gaditana • Cloning of 4 PTS1 predicted genes from N. gaditana • Subcellular localization of predicted PTS1 proteins by fluorescence microscopy • Genomic transformation of Nannochloropsis sp. • Cloning of inducible Nannochloropsis promoters
  • 15. Methods Cloning of N. gaditana genes predicted to have PTS1 • Cloning of CDS into pJET1.2 cloning vector – 3 Full-length CDS of single exon genes; – 1 C-terminal exon. • Sequence analysis • Sub-cloning of insert from pJET1.2 into plant expression vector pCAT_EYFP.
  • 16. Methods Subcellular localization of predicted PTS1 Proteins 1. Coating gold particles with DNA – 40 ng/μl plasmid DNA – 50 μl 2.5 M CaCl2 – 20 μl 0.1 M spermidine 2. Transient expression in onion epidermal cells – Biolistic transformation using a gene gun – 12 h dark incubation 3. Microscopy – Inverted fluorescence microscope (Nikon TE 2000u)
  • 17. Methods Genomic transformation of Nannochloropsis • By electroporation • Using pSelect100 vector – Linearized by BamHI – 2 antibiotic resistance genes: Hygromycin B and Ampicillin Based on the method described in Vieler et. al. (2012) pSELECT100 (C. Benning lab)
  • 18. Methods Cloning of inducible Nannochloropsis promoters • Modification of pSELECT100 vector • Making an expression vector for N. oceanica • Making a cloning vector for N. gaditana Extended pSELECT vector with 2nd expression cassette (Reumann lab) Pre-existing pSELECT vector (Benning lab)
  • 19. Results PCR amplification of Nannochloropsis gaditana genes • Ng_AroDH: 2355bp • Ng_GOX: 750 bp • Ng_ EAP: 1458 bp • Ng_AOX: 2253 bp
  • 20. Results 2.2.Vectors 2.2.1.pCAT The pCAT is a plant expression vector. Which localization experiments in the Reumann lab (Lingner used in this study had inserted EYFP sequence CamV 35S promoter. The known A. thaliana PTS1 EYFP-coding gene. Such construct pCAT-EYFP-protein which will be targeted to the peroxisomes. peroxisomal Cloning of 4 N. gaditana genes into the pJET vector • Ng_AroDH: 2355bp • Ng_GOX: 750 bp • Ng_ EAP: 1500 bp • Ng_AOX: 2253 bp inserted 2.2.2.The developed Cloning (www.molecular-cloning/ Figure (ApR)-eco47IR-cloning (www.thermoscientificbio.clonejet-Transformed in E. Coli JM 109
  • 21. Results PCR and restriction digestion analysis of Plasmid Plasmid analysis of inserts in pJET vector • Ng_AroDH: 2355bp • Ng_GOX: 750 bp • Ng_ EAP: 1458 bp • Ng_AOX: 2253 bp
  • 22. • Positive plasmids were sent for sequencing. • Sequencing result analyzed by blast and multiple alingments • Resulting seqeunce was checked for PTS1 tripeptides. • Sequencing result was also checked for gene specific primer. Results Sequence analysis of the insert in pJet vector
  • 23. Results Sub-cloning of CDS/C-terminal exons in the back of EYFP in EYFP/pCAT vector • Restriction digestion of insert and vector pCAT • Ligation of digested sticky fragments • Analysis of colonies by colonies PCR • Analysis of plasmids by PCR and digestion pCAT_EYFP_DECR: 4200 bp DECR: 1000 bp Analytical digestion of the destination vector EYFP-DECR/ pCAT
  • 24. Results E coli colonies transformed with EYFP-Ng_EAP/pCAT on LB ampicillin plates Digestion and PCR analysis of plasmids, EYFP-Ng_EAP/pCAT and EYFP-Ng_GOX/pCAT E coli colonies transformed with EYFP-Ng_ EAP/pCAT on LB ampicillin plates • Ng_GOX: 750 bp • Ng_ EAP: 1458 bp
  • 25. Subcellular localization studies by transient expression in onion cells Experimental validation of predicted PTS1 by in vivo subcellular targeting analysis via transient expression in onion cells
  • 26. Results Subcellular localization of selected Nannochloropsis gaditana proteins carrying predicted PTS1 tripeptides Int. # Annotation Predicte d PTS1 Acronym Length In vivo subcellular targeting P2 Arogenate dehydrogenase PKL> Ng_AroDH 785 aa Plasmid not obtained P3 Peroxisomal glycolate oxidase SKI> Ng_GOX 390 aa Peroxisomes P8 embryogenesis-associated protein emb8 SRL> Ng_EAP 486 aa Peroxisomes P9 acyl- oxidase ARL> Ng_AOX 751 aa Peroxisomes
  • 27. Result Genomic transformation of N. oceanica CCMP1779 27 N. Oceanica ½ salinity f/2 agar plates containing 50 μg/ml Hygromycin B Positive control Negative control
  • 28. Results Subcloning of inducible Nannochloropsis promoters • The pSELECT plasmid obtained from Prof. Dr. C. Benning contains only a single expression cassette. • 2nd expression cassette: For subcellular localization studies (EYFP fusions) and to overexpress genes of interest in N. oceanica.
  • 29. Results Subcloning of inducible Nannochloropsis promoters • Amplification of lipid droplet surface proteins from pSELECT 100 and N. gaditana • Cloning of PCR product into pJET Vector • Sub-cloning of the LDSP promoter from pJET into pSELECT100 Preparative PCR to amplify LDSP promoter from pSELECT100 and N. gaditana PRO_Noce_LDSP: 750 bp PRO_Nagd_LDSP: 1000 bp
  • 30. Results Subcloning of inducible Nannochloropsis promoters E. coli transformed with PRO_Ngad_LDSP/pJET (B) and PRO_Noce_LDSP/pJET (A) colonies on LB ampicillin plates Plasmids analysis of the insert in pJET vector PRO_Noce_LDSP: 750 bp PRO_Nagd_LDSP: 1000 bp
  • 31. Subcloning of inducible Nannochloropsis promoters E. coli transformed with Plasmid analysis of PRO_Noce_LDSP/pSELECT10 PRO_Noce_LDSP/pSELECT100 colonies on LB ampicillin plates
  • 32. Manual analysis searching was done for the KpnI site of the forward subcloning primer (GTAGGTACC GAGGTCC TGTTTGGATT TTTTGCT) and the ClaI site of the reverse primer (CAGAAACTCT ATCTCAAGAT AAG ATCGATGCA):
  • 33. Result Sequencing analysis of PRO_Noce_LDSP/pSELECT100 Electropherogram read using Finch TV program
  • 34. Summary • 3 predicted PTS1 proteins of interest from Nannochloropsis gaditana were sub-cloned into pJET1.2: Ng_GOX, Ng_EAP and Ng_AOX was successful while Ng_ADH was not successful. • Cloned genes were sub-cloned into the plant expression vector pCAT. • The 3 predicted PTS1 proteins were localized to peroxisomes in onion epidermal cells. • Reproduction of nuclear transformation of Nannochloropsis was successfully performed • The LDSP promoter from pSELECT was cloned into pJET vector and sub-cloned into MCS of pSELECT100 • LDSP from Nannochloropsis gaditana successfully cloned into pJET1.2
  • 35. Future work 1. Sub clone N. gaditana LDSP Promoter from pJET1.2 into pSELECT 2. Reproduction of nuclear transformation of N. gaditana with plasmids from Posewitz (plasmids obtained from the Posewitz lab) 3. Further construction of 2nd expression cassette in pSELECT
  • 36. Acknowledgements • Prof. S. Reumann • Manish Budathoki • Dmitry Kechasov • Dr. G. Chowdhary • Eli Drange Vee • All lab members • University of Stavanger

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