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Analysis of Peroxisomal Lipid Metabolism in the Oleaginous Microalga Nannochloropsis and Development of Synthetic Biology Tools for Genetic Engineering
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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 TechnologyDepartment of Mathematics and Natural
SciencesUniversity of Stavanger
20. August 2014
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 (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
Introduction
Introduction
• Grow fast• High biofuel yields• Consume CO2
• Do not compete with agriculture• Microalgal biomass can be used for fuel, feed and food • Purify wastewater
Why algae
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)
Introduction
• 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)
Nannochloropsis sp.
EPA: Eicosapentaenoic acid.TAG: Triacylglycerol
Why Nannochloropsis gaditana?
Introduction
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)
Introduction
• 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.
Development of Algal Industry
Introduction
• 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.
Algal lipids
Introduction
• 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)
Peroxisomes
Anatomy of peroxisome.UNSW Cell Biology, 2008(http://cellbiology.med.unsw.edu.au/units/science/lecture0806.htm)
Small (0.5-1 µm), single membrane-bound organelles
Introduction
• 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.
Synthetic Biology
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
Introduction
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.
Methods
• 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
Approaches used
Methods
• 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.
Cloning of N. gaditana genes predicted to have PTS1
Methods
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)
Subcellular localization of predicted PTS1 Proteins
Methods
• 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)
Genomic transformation of Nannochloropsis
pSELECT100 (C. Benning lab)
Methods
• Modification of pSELECT100 vector• Making an expression vector for N. oceanica• Making a cloning vector for N. gaditana
Cloning of inducible Nannochloropsis promoters
Extended pSELECT vector with 2nd expression cassette(Reumann lab)
Pre-existing pSELECT vector(Benning lab)
Results
PCR amplification of Nannochloropsis gaditana genes
• Ng_AroDH: 2355bp• Ng_GOX: 750 bp• Ng_ EAP: 1458 bp • Ng_AOX: 2253 bp
Results
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
Transformed in E. Coli JM 109
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
• 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
Results
• 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
Sub-cloning of CDS/C-terminal exons in the back of EYFP in EYFP/pCAT vector
Analytical digestion of the destination vector EYFP-DECR/ pCAT
pCAT_EYFP_DECR: 4200 bpDECR: 1000 bp
Results
E coli colonies transformed with EYFP-Ng_EAP/pCAT on LB ampicillin platesDigestion 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
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
Results
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
Subcellular localization of selected Nannochloropsis gaditana proteins carrying predicted PTS1 tripeptides
Result
Genomic transformation of N. oceanica CCMP1779
27
½ salinity f/2 agar plates containing 50 µg/ml Hygromycin B
N. Oceanica Negative controlPositive control
Results
• 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.
Subcloning of inducible Nannochloropsis promoters
Results
• 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
Subcloning of inducible Nannochloropsis promoters
Preparative PCR to amplify LDSP promoter from pSELECT100 and N. gaditana
PRO_Noce_LDSP: 750 bpPRO_Nagd_LDSP: 1000 bp
Results
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 bpPRO_Nagd_LDSP: 1000 bp
Subcloning of inducible Nannochloropsis promoters
Plasmid analysis of PRO_Noce_LDSP/pSELECT10E. coli transformed with PRO_Noce_LDSP/pSELECT100 colonies on LB ampicillin plates
Subcloning of inducible Nannochloropsis promoters
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):
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_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
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
Acknowledgements
• Prof. S. Reumann• Manish Budathoki• Dmitry Kechasov • Dr. G. Chowdhary• Eli Drange Vee• All lab members
• University of Stavanger