UNIVERSITI PUTRA MALAYSIA

VU THI QUYNH CHI

FBSB 2013 2

ESTABLISHMENT OF NUCLEAR TRANSFORMATION PROTOCOL OF GREEN MICROALGAE Ankistrodesmus convolutus

USING ELECTROPORATION

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ESTABLISHMENT OF NUCLEAR TRANSFORMATION PROTOCOL OF GREEN MICROALGAE Ankistrodesmus convolutus

USING ELECTROPORATION

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

By

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

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The thesis was dedicated to

my beloved husband, Dr. Tran Thanh

my parents, Mr. Vu Van Tro and Mrs. Nong Thi Ngoc

my mother-in-law, Mrs. Nguyen Thi Hoan

my brothers and sisters

my nieces and nephews

for their endless love and sacrifice as well as their encouragement which

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

ESTABLISHMENT OF NUCLEAR TRANSFORMATION PROTOCOL OF GREEN MICROALGAE ANKISTRODESMUS CONVOLUTUS USING

ELECTROPORATION

By

VU THI QUYNH CHI

February 2013

Chair: Assoc Professor Suhaimi B Napis, PhD

Faculty: Biotechnology and Biomolecular Sciences

Despite the availability of some expression systems such as mammalian cell,

yeast, bacteria, insect cell, and fungi, green microalgae have risen as

interesting alternative systems for the expression of recombinant proteins. The

world of algae is tremendously diverse but the number of species exploited for

biotechnological applications is quite limited due to the lack of transformation

system and molecular information. As a green microalgae, Ankistrodesmus

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

driven by double CaMV35S promoter. Minimal inhibitory concentration test of

hygromycin showed that non-transformed A. convolutus was inhibited at the

concentration of 40 mg/L in solid medium after 10 days of inoculation. The

parameters of electroporation including cell treatment, electric pulse,

concentration of expression vector, concentration of carrier DNA (salmon sperm

DNA) and cuvette width were optimized. The results of this study showed that

highest transformation efficiency of A. convolutus was obtained when usingcells

treated with cellulase (2%) and pectinase (0.3%) before transformation and

electric pulse of 1800 V. The optimal concentrations of the expression vector

and carrier DNA were10 µg/mL and 50 µg/mL, respectively. Electroporation

cuvette with 4 mm gap was more efficient than the 2 mm gap cuvette in

introducing foreign gene into A. convolutus. The highest transformation

efficiency was 481 transformants per µg DNA use and the transformation yield

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

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

feasible to exploit A. convolutus as an alternative system for the expression of

recombinant proteins. Particularly, the prospective of using transgenic A.

convolutus as oral vaccine or source of recombinant lutein for poultry can be

examined.

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Abstract tesis yang dikemukakan kepada Senat Universiti Putra Malaysia

sebagai memenuhi keperluan untuk ijazah Master di Sains

PENYEDIAAN PROTOKOL TRANSFORMASI NUKLEAR UNTUK MIKROALGAE HIJAU ANKISTRODESMUS CONVOLUTUS

MENGGUNAKAN KAEDAH ELEKTROPORASI

Oleh

VU THI QUYNH CHI

Februari 2013

Pengerusi : Profesor Madya Suhaimi B Napis, PhD Fakulti: Bioteknologi dan Sains Biomolekul

Walaupun terdapat beberapa jenis sistem ekpresi gen seperti sel mamalia, yis,

bakteria, sel serangga and kulat, mikroalgae hijau telah dibangunkan sebagai

sistem alternatif yang menarik untuk mengekpreskan rekombinan protein.

Dunia algae adalah penuh dengan kepelbagaian tetapi bilangan spesies yang

dieksploitasikan untuk bioteknologi adalah agak terhad disebabkan kekurangan

sistem transformasi dan maklumatbiologi molekul. Ankistrodesmus convolutus

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

phosphotransferase (hpt) yang membolehkan rintangan terhadap hygromycin

dikawalatur oleh promoter CaMV35S yang berganda. Ujian kepekatan

perencatan minimal terhadap hygromycin menunjukkan bahawa A. convolutus

yang tidak ditransformasikan direncat pada kepekatan 40 mg/L dan 20 mg/L

dalam media pepejal BBM selepas hari 10 dan dalam media cecair BBM setelah

hari 5 masing-masing. Parameter untuk elektroporasi termasuk rawatan sel,

kadar arus elektrik, kepekatan vektor pengekspresan, kepekatan DNA pembawa

DNA (sperma ikan salmon) dan lebar kuvet telah dioptimumkan. Keputusan

untuk kajian ini menunjukkan kecekapan transformasi yang tertinggi telah

didapati menggunakan sel yang dirawat dengan selulase (2%) dan pektinase

(0.3%) sebelum transformasi dan kadar arus elektrik pada 1800V. Kepekatan

optima untuk vektor ekpresi dan pembawa DNA adalah 10 µg/mL dan 50

µg/mL, masing-masing. Kuvet elektroporasi dengan lebar 4 mm adalah lebih

sesuai berbanding dengan kuvetlebar 2 mm dalam memperkenalkan gen asing

kedalam A. convolutus. Kecekapan transformasi yang tertinggi adalah 481

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

menunjukkan integrasi gusA kedalam genom pada sel yang ditransformasikan.

Kajian ini telah berjaya memperkenalkan gen asing kedalam A. convolutus

melalui kaedah elektroporasi. Maka,kejayaan ini dapat memudahkan

pengekspresan gen asing dalam spesis algae ini. Penemuan ini memungkinkan

untuk mengeksploitasikan A. convolutus sebagai sistem alternatif untuk

pengekspresan protein rekombinan. Secara khususnya, kaedah yang

dibangunkanakan berpotensi untukmenjadikan A. convolutus transgenik

sebagai vaksin oral atau sumber lutein rekombinan untuk penternakan mampu

dikaji.

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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

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

SitiNor Akmar Abdullah for their kind suggestion and encouragement

throughout the study.

My special thanks are given to the Director Board of Rubber Research of

Vietnam and the Director Board of GIM 777 Bhd. Sdn (Malaysia), especially Mr.

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

of Vietnam for their encouragement and support during my study period.

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,

Dr. Huynh Ky, Dr. Le Vinh Thuc, Dr. Shahanaz Sultana, Mrs. Samanthi for their

technical knowledge and support. Many thanks go to my dear labmates for their

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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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:

Suhaimi Napis, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Raha Abdul Rahim, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member) Hishamuddin Omar, PhD Lecturer Faculty of Sciences Universiti Putra Malaysia (Member) Datin Siti Nor Akmar Abdullah, PhD

Asscociate Professor Faculty of Agriculture Universiti Putra Malaysia (Member)

BUJANG BIN KIM HUAT, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia

Date:

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DECLARATION

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

Date: 27th February 2013

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TABLE OF CONTENTS

ABSTRACT iii

ABSTRAK vi

ACKNOWLEDGEMENTS ix

APPROVAL x

DECLARATION xii

LIST OF TABLES xvi

LIST OF FIGURES xvii

LIST OF ABBREVIATIONS xviii

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW 4

2.1. Definition and diversity of algae 4

2.2. Green microalgae Ankistrodesmus convolutus Corda. 9

2.3. Potentials of green microalgae 12

2.4. Green microalgae as alternative systems for expression of recombinant proteins 16

2.5. Methods of gene transformation into microalgae 24

2.5.1.Glassbead/silicon carbide whisker 25

2.5.2.Electroporation 26

2.5.3.Agrobacterium-mediated transformation 27

2.5.4.Particle bombardment 28

2.6. Selective markers and reporter genes in microalgae transformation 29

3 MATERIALS AND METHODS 32

3.1. Construction of expression vector 32

3.2. Preparation of Escherichia coli DH5α 33

3.3. Bacterial transformation 34

3.4. Plasmid DNA preparation 34

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3.5. DNA quality and quantitation 36

3.6. Agarose gel electrophoresis 36

3.7. Algae culture 37

3.8. Determination of the minimal inhibitory concentration of hygromycin on the growth of A. convolutus 37

3.9. Optimization of electroporation parameters 38

3.9.1.Chemicals and consumables 38

3.9.2.Preparation of algae cells 38

3.9.3.Preparation of electroporation buffer 39

3.9.4.Optimization of electroporation protocols 39

3.9.5.Data analysis 41

3.9.6.Molecular analysis of putative transgenic A. convolutus transformants 41

3.9.6.1.Genomic DNA extraction from transgenic A. convolutus 41

3.9.6.2.Screening of transgenic A. convolutus by PCR 42

3.9.6.3.Southern blot analysis of transgenic A. convolutus 43

4 RESULTS 47

4.1. Construction of pEXP-GUS vector 47

4.2. Determination of minimal inhibitory concentration of hygromycin for wild type A. convolutus 49

4.3. Optimization of electroporation parameters for transformation of A. convolutus 53

4.3.1. Effects of cell-wall degrading enzymes on the transformation efficiency of A. convolutus 53

4.3.2. Effects of electric pulse on the transformation efficiency of A. convolutus 55

4.3.3. Effects of expression vector concentration on the transformation efficiency of A. convolutus 59

4.3.4. Effects of carrier DNA concentration on the transformation efficiency of A. convolutus 59

4.3.5. Effects of cuvette width on the transformation efficiency of A. convolutus 60

4.4. Molecular analysis of A. convolutus by PCR and Southern blot analysis 61

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5 DISCUSSION 67

6 SUMMARY, CONCLUSION AND RECOMMENDATIONS FOR FUTURE RESEARCH 77

6.1. Conclusion 77

6.2. Future research 78

REFERENCES 80

APPENDICES 92

BIODATA OF STUDENT 98