UNIVERSITI PUTRA MALAYSIA
ESTABLISHMENT OF NUCLEAR TRANSFORMATION PROTOCOL OF
GREEN MICROALGAE Ankistrodesmus convolutus
USING ELECTROPORATION
VU THI QUYNH CHI
FBSB 2013 2
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ESTABLISHMENT OF NUCLEAR
TRANSFORMATION PROTOCOL OF GREEN
MICROALGAE Ankistrodesmus convolutus
USING ELECTROPORATION
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VU THI QUYNH CHI
MASTER OF SCIENCE
UNIVERSITI PUTRA MALAYSIA
2013
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ESTABLISHMENT OF NUCLEAR TRANSFORMATION PROTOCOL OF
GREEN MICROALGAE Ankistrodesmus convolutus
USING ELECTROPORATION
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By
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VU THI QUYNH CHI
Thesis submitted to the School of Graduate Studies, Universtiti Putra
Malaysia, in Fulfilment of the Requirements for the
Degree of Master of Science
February 2013
my beloved husband, Dr. Tran Thanh
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The thesis was dedicated to
my parents, Mr. Vu Van Tro and Mrs. Nong Thi Ngoc
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my mother-in-law, Mrs. Nguyen Thi Hoan
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my nieces and nephews
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my brothers and sisters
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for their endless love and sacrifice as well as their encouragement which
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led me to this achievement.
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in
fulfillment of the requirement for the degree of Master of Science
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February 2013
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VU THI QUYNH CHI
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ESTABLISHMENT OF NUCLEAR TRANSFORMATION PROTOCOL OF
GREEN MICROALGAE ANKISTRODESMUS CONVOLUTUS USING
ELECTROPORATION
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Chair: Assoc Professor Suhaimi B Napis, PhD
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Faculty: Biotechnology and Biomolecular Sciences
Despite the availability of some expression systems such as mammalian cell,
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yeast, bacteria, insect cell, and fungi, green microalgae have risen as
interesting alternative systems for the expression of recombinant proteins. The
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world of algae is tremendously diverse but the number of species exploited for
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biotechnological applications is quite limited due to the lack of transformation
system and molecular information. As a green microalgae, Ankistrodesmus
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convolutus is a fast growing green microalgae species that contains appreciable
amount of carotenoids, polyunsaturated fatty acids and lutein which make this
speciesapotential feed in poultry industry. Development of a transformation
protocol for this species is essential prior to any of its application for gene
manipulation. This study was aimed to establish a nuclear transformation
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protocol for A. convolutus using electroporation method. The expression vector,
pEXP-GUS was created by the modification of pMDC-141 vector. This
expressionvectorharbors
a
β-glucoronidase
encoding
gene
(gusA)
and
ahygromycin phosphotransferase gene (hpt) conferringhygromycin resistance
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driven by double CaMV35S promoter. Minimal inhibitory concentration test of
hygromycin showed that non-transformed A. convolutus was inhibited at the
parameters
of
electroporation
including
cell
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concentration of 40 mg/L in solid medium after 10 days of inoculation. The
treatment,
electric
pulse,
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concentration of expression vector, concentration of carrier DNA (salmon sperm
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DNA) and cuvette width were optimized. The results of this study showed that
highest transformation efficiency of A. convolutus was obtained when usingcells
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treated with cellulase (2%) and pectinase (0.3%) before transformation and
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electric pulse of 1800 V. The optimal concentrations of the expression vector
and carrier DNA were10 µg/mL and 50 µg/mL, respectively. Electroporation
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cuvette with 4 mm gap was more efficient than the 2 mm gap cuvette in
introducing foreign gene into A. convolutus. The highest transformation
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efficiency was 481 transformants per µg DNA use and the transformation yield
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was 48 x 10-6 cells. A total of 14 transformants were obtained after six rounds
of subculturing. The presence of transgenes including hpt gene and gusA gene
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were then analyzed by PCR and Southern blot analysis. The resultsrevealed the
presence of both hpt gene and gusA gene in three transformants namely AcG3,
AcG4, AcG14. Among these three A. convolutus transformants, only one
transformants gave hybridization signal when the XbaI-digested genomic DNA
of PCR-positive transformants was hybridized to a biotin-labelled gusA gene-
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specific probe. This result implied the integration of gusA into the genomes of
the transformed cells. The present study was successful in introducing the
foreign genes into A. convolutus using electroporation. Hence,this success
facilitates the expression of foreign genes in this algae species. It is now
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feasible to exploit A. convolutus as an alternative system for the expression of
recombinant proteins. Particularly, the prospective of using transgenic A.
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convolutus as oral vaccine or source of recombinant lutein for poultry can be
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examined.
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Abstract tesis yang dikemukakan kepada Senat Universiti Putra Malaysia
sebagai memenuhi keperluan untuk ijazah Master di Sains
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PENYEDIAAN PROTOKOL TRANSFORMASI NUKLEAR UNTUK
MIKROALGAE HIJAU ANKISTRODESMUS CONVOLUTUS
MENGGUNAKAN KAEDAH ELEKTROPORASI
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Oleh
VU THI QUYNH CHI
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Februari 2013
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Pengerusi : Profesor Madya Suhaimi B Napis, PhD
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Fakulti: Bioteknologi dan Sains Biomolekul
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Walaupun terdapat beberapa jenis sistem ekpresi gen seperti sel mamalia, yis,
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bakteria, sel serangga and kulat, mikroalgae hijau telah dibangunkan sebagai
sistem alternatif yang menarik untuk mengekpreskan rekombinan protein.
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Dunia algae adalah penuh dengan kepelbagaian tetapi bilangan spesies yang
dieksploitasikan untuk bioteknologi adalah agak terhad disebabkan kekurangan
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sistem transformasi dan maklumatbiologi molekul. Ankistrodesmus convolutus
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adalah sejenis spesis mikroalgae hijau yang tumbuh dengan cepat dan
mengandungi jumlah karotenoid, asid lemak poli tak tepu dan lutein yang
ketara menjadikan spesies ini berpotensi diganakan sebagai makanan didalam
industri penternakan haiwanPenyediaan protokol transformasi untuk spesies ini
adalah penting untuk sebarang aplikasi bagi manipulasi gen. Kajian ini adalah
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bertujuan untuk membangunkan protokol transformasi untuk A. convolutus
menggunakan kaedah elektroporasi. Vektor ekspresi, pEXP-GUS
telah
dihasilkan melalui pengubahsuaian vector pMDC 141. Vektor ekspresi ini
membawa gen pengekod β-glucoronidase (gusA) dan gen hygromycin
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phosphotransferase (hpt) yang membolehkan rintangan terhadap hygromycin
dikawalatur oleh promoter CaMV35S yang berganda. Ujian kepekatan
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perencatan minimal terhadap hygromycin menunjukkan bahawa A. convolutus
yang tidak ditransformasikan direncat pada kepekatan 40 mg/L dan 20 mg/L
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dalam media pepejal BBM selepas hari 10 dan dalam media cecair BBM setelah
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hari 5 masing-masing. Parameter untuk elektroporasi termasuk rawatan sel,
kadar arus elektrik, kepekatan vektor pengekspresan, kepekatan DNA pembawa
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DNA (sperma ikan salmon) dan lebar kuvet telah dioptimumkan. Keputusan
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untuk kajian ini menunjukkan kecekapan transformasi yang tertinggi telah
didapati menggunakan sel yang dirawat dengan selulase (2%) dan pektinase
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(0.3%) sebelum transformasi dan kadar arus elektrik pada 1800V. Kepekatan
optima untuk vektor ekpresi dan pembawa DNA adalah 10 µg/mL dan 50
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µg/mL, masing-masing. Kuvet elektroporasi dengan lebar 4 mm adalah lebih
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sesuai berbanding dengan kuvetlebar 2 mm dalam memperkenalkan gen asing
kedalam A. convolutus. Kecekapan transformasi yang tertinggi adalah 481
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transforman untuk setiap µg DNA yang digunakan dan hasil transformasi adalah
48 x 10-6 sel untuk A. convolutus. Sejumlah 14 transforman telah diperolehi
setelah enam pusingan sub-kulturan. Pengekspresan transgen gen hpt dan gen
gusAtelah dianalisa dengan kaedah PCR dan Southern blot. Keputusan
menunjukkan kehadiran untuk kedua-dua gen hpt dan gen gusA dalam tiga
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transforman yang dinamakan AcG3, AcG4, AcG14. Antara tiga transforman A.
convolutus ini, hanya satu transforman memberi isyarat hibridisasi apabila
genomik DNA transforman positif-PCR yang telah dipotong dengan enzim XbaI
dihibridisasi kepada probe spesifik yang dilabelkan dengan biotin. Keputusan ini
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menunjukkan integrasi gusA kedalam genom pada sel yang ditransformasikan.
Kajian ini telah berjaya memperkenalkan gen asing kedalam A. convolutus
kaedah
elektroporasi.
Maka,kejayaan
ini
dapat
memudahkan
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melalui
pengekspresan gen asing dalam spesis algae ini. Penemuan ini memungkinkan
dibangunkanakan
protein
rekombinan.
berpotensi
Secara
khususnya,
kaedah
yang
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pengekspresan
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untuk mengeksploitasikan A. convolutus sebagai sistem alternatif untuk
untukmenjadikan
A. convolutus transgenik
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dikaji.
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sebagai vaksin oral atau sumber lutein rekombinan untuk penternakan mampu
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ACKNOWLEDGEMENTS
First of all, I would like to express my gratitude to my supervisor, Assoc. Prof.
Dr. Suhaimi Napis for giving me the opportunity to pursue my studies in the
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field of Biotechnology. My gratitudeis given to Dr. Hishamuddin Omar. His
guidance and encouragement stimulated me to work in this project. My sincere
appreciation also goes to Prof. Raha Abdul Rahim and Assoc. Prof. Dr. Datin
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SitiNor Akmar Abdullah for their kind suggestion and encouragement
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throughout the study.
My special thanks are given to the Director Board of Rubber Research of
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Vietnam and the Director Board of GIM 777 Bhd. Sdn (Malaysia), especially Mr.
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Don Tham for the financial support during my study in Malaysia. I also want to
extend my gratitude to my beloved colleagues in the Rubber Research Institute
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of Vietnam for their encouragement and support during my study period.
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My sincere thanks also go to Mr. Noroozi Mostafa for his kind help and support
during the study. I would like to extend my gratitude to Dr. Nguyen Anh Nghia,
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Dr. Huynh Ky, Dr. Le Vinh Thuc, Dr. Shahanaz Sultana, Mrs. Samanthi for their
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technical knowledge and support. Many thanks go to my dear labmates for their
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friendship, support and encouragement.
Last but not least,I wish to express my deepest gratitude and appreciation to
my parents, siblings and family members, especially my husband Mr Tran
Thanh, for their endless love and constant support throughout my study which
led me to this achievement.
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Suhaimi Napis, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Chairman)
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Hishamuddin Omar, PhD
Lecturer
Faculty of Sciences
Universiti Putra Malaysia
(Member)
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Raha Abdul Rahim, PhD
Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Member)
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This thesis was submitted to the Senate of Universiti Putra Malaysia and has
been accepted as fulfillment of the requirement for the degree of Master of
Science. The members of the Supervisory Committee were as follows:
BUJANG BIN KIM HUAT, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
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Datin Siti Nor Akmar Abdullah, PhD
Asscociate Professor
Faculty of Agriculture
Universiti Putra Malaysia
(Member)
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DECLARATION
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I declare that the thesis is my original work except for quotations and citations
which have been duly acknowledged. I also declare that it has not been
previously, and is not concurrently, submitted for any other degree at Universiti
Putra Malaysia or at any other institution.
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VU THI QUYNH CHI
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Date: 27th February 2013
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TABLE OF CONTENTS
ABSTRACT
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ABSTRAK
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ACKNOWLEDGEMENTS
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APPROVAL
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DECLARATION
LIST OF TABLES
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LIST OF FIGURES
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LIST OF ABBREVIATIONS
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CHAPTER
1
4
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2 LITERATURE REVIEW
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1 INTRODUCTION
2.3. Potentials of green microalgae
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2.4. Green microalgae as alternative systems for expression of
recombinant proteins
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2.5. Methods of gene transformation into microalgae
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2.2. Green microalgae Ankistrodesmus convolutus Corda.
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2.1. Definition and diversity of algae
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2.5.2.Electroporation
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2.5.1.Glassbead/silicon carbide whisker
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2.5.4.Particle bombardment
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2.5.3.Agrobacterium-mediated transformation
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2.6. Selective markers and reporter genes in microalgae transformation 29
3 MATERIALS AND METHODS
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3.1. Construction of expression vector
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3.2. Preparation of Escherichia coli DH5α
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3.3. Bacterial transformation
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3.4. Plasmid DNA preparation
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3.5. DNA quality and quantitation
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3.6. Agarose gel electrophoresis
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3.7. Algae culture
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3.8. Determination of the minimal inhibitory concentration of hygromycin
on the growth of A. convolutus
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3.9. Optimization of electroporation parameters
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3.9.2.Preparation of algae cells
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3.9.1.Chemicals and consumables
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3.9.3.Preparation of electroporation buffer
3.9.4.Optimization of electroporation protocols
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3.9.5.Data analysis
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3.9.6.Molecular analysis of putative transgenic A. convolutus
transformants
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3.9.6.1.Genomic DNA extraction from transgenic
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A. convolutus
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3.9.6.3.Southern blot analysis of transgenic A. convolutus
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3.9.6.2.Screening of transgenic A. convolutus by PCR
RESULTS
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4.2. Determination of minimal inhibitory concentration of hygromycin
for wild type A. convolutus
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4.1. Construction of pEXP-GUS vector
4.3. Optimization of electroporation parameters for transformation of
A. convolutus
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4.3.1. Effects of cell-wall degrading enzymes on the transformation
efficiency of A. convolutus
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4.3.2. Effects of electric pulse on the transformation efficiency of
A. convolutus
4.3.3. Effects of expression vector concentration on the
transformation efficiency of A. convolutus
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4.3.4. Effects of carrier DNA concentration on the transformation
efficiency of A. convolutus
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4.3.5. Effects of cuvette width on the transformation efficiency of
A. convolutus
4.4. Molecular analysis of A. convolutus by PCR and Southern blot
analysis
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DISCUSSION
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SUMMARY, CONCLUSION AND RECOMMENDATIONS FOR FUTURE
RESEARCH
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6.2. Future research
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6.1. Conclusion
REFERENCES
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APPENDICES
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BIODATA OF STUDENT
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