USWATUN BAHIRAH BT AHMAD HISHAM UNIVERSITI SAINS MAL

45 Pages·2012·0.59 MB·English

CHARACTERIZATION OF A NEW
BACTERIAL VIRUS FROM ORANGUTAN
(Pongo pygmaeus)

USWATUN BAHIRAH BT AHMAD HISHAM

UNIVERSITI SAINS MALAYSIA

2015
CHARACTERIZATION OF A NEW
BACTERIAL VIRUS FROM ORANGUTAN
(Pongo pygmaeus)

by

USWATUN BAHIRAH BT AHMAD HISHAM

Thesis submitted in fulfillment of the requirements
for the degree of
Master of Science

March 2015
ACKNOWLEDGEMENT
In the name of Allah, The Most Gracious and The Most Merciful
Prayers and peace be upon His Prophet Muhammad S.A.W.
Foremost, I would like to express the deepest appreciation to my supervisor,
Assoc. Prof. Dr. Yahya Mat Arip for his exemplary guidance and constant motivation
throughout this project. His continuous support, patience and knowledge will always be
remembered. I am grateful for his effort and time despite of his extraordinarily busy
schedule.
I express warm thanks to all postgraduate students from Lab 218 for all guidance
and advices. It is through their kindness and encouragement that I have gained and
grown. To all lab assistants and all members of the Electron Microscope Unit staffs, I
will always appreciate your helps and your cooperation upon completion of this project.
To the Ministry of Higher Education and USM, I would like to take this
opportunity to thank you for your generosity in funding this project. I sincerely appreciate
the Institute of Postgraduate Studies USM for granted me the USM Fellowship.
I am highly indebted to my beloved parents and family for their endless love,
blessings and encouragement. I will always love and treasure them forever. A special
thanks to my beloved sister, Hanis Shaheera Bt. Ahmad Hisham for being there when I
need it the most. To my fiancé, Mohd Ashraf B. Abdul Rahman, thank you for your
prayers. For all who has contributed to this project, I owe you a heartfelt appreciation.
Thank you.
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TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENTS ii
TABLE OF CONTENTS iii
LIST OF TABLES viii
LIST OF FIGURES ix
LIST OF ABBREVIATIONS xi
LIST OF APPENDICES xv
LIST OF SYMBOLS xvi
ABSTRAK xvii
ABSTRACT xix

CHAPTER 1: INTRODUCTION 1

CHAPTER 2: LITERATURE REVIEW 3
2.1 Bacteriophages 3
2.2 History of bacteriophages 4
2.3 Structure of bacteriophages 5
2.3.1 Basic architecture of bacteriophages 5
2.3.2 Basic morphological types of bacteriophages 6
2.3.3 Bacteriophage T4 8
2.4 Evolution of bacteriophages 10
2.4.1 Mosaicsm of phage DNA and non-homologous 10
recombination
2.4.2 Homologous recombination 11
2.5 Diversity of bacteriophages 12
2.6 Classification of bacteriophages 14
2.6.1 History of bacteriophages classification 14
2.6.2 Criteria of bacteriophages classification 15
2.6.3 The families of bacteriophages 15
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2.7 Mechanisms of phage infections 20
2.7.1 The lytic cycle 22
2.7.2 The lysogenic cycle 24
2.8 Application of bacteriophages 26
2.8.1 Phage display in nanotechnology for antibody engineering 27
2.8.2 Phage as an antimicrobial tool 27
2.8.3 Phage as gene-delivery 29
2.9 The microbiota in orangutan 29

CHAPTER 3: MATERIALS AND METHODS 30
3.1 Materials 31
3.2 Preparation of media 31
3.2.1 Luria-Bertani (LB) agar 31
3.2.2 Luria-Bertani (LB) broth 31
3.2.3 Half Luria-Beetani (1/ LB) agar 31
2
3.2.4 Luria-Bertani top agar 31
3.2.5 LB-Ampicillin agar 32
3.2.6 LB-Kanamycin agar 32
3.3 Preparation of working solutions 32
3.3.1 Tris-Magnesium-Sodium (TMS) chloride buffer 32
3.3.2 3M Sodium acetate (NaOAc), pH 5.2 33
3.3.3 40% of glycerol stock 33
3.3.4 1X Tris-Borate-EDTA (TBE) buffer 33
3.3.5 1% (w/v) agarose gel 33
3.3.6 Isopropyl β-D-1-thiogalactopyranoside (IPTG) 34
3.3.7 Ampicillin solution (100mg/ml) 34
3.3.8 Kanamycin solution (100mg/ml) 34

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3.3.9 Stock solutions for sodium dodecyl sulphate polyacrylamide 34
gel electrophoresis (SDS-PAGE)
3.4 Host and phage strain, competent cells and cloning vector 35
3.5 Nucleotide sequences of the primers used in PCR analysis 39
3.6 Verification of phage UB1 stock 39
3.6.1 Propagation of phage UB1 in E. coli O157:H7 39
3.6.2 Purification of phage UB1 stock 40
3.6.3 Enrichment of phage UB1 stock 41
3.6.4 Morphological study of phage UB1 41
3.7 Host specificity of phage UB1 41
3.8 Viability of phage UB1 stock at different physicochemical 42
attributes
3.8.1 pH 42
3.8.2 Temperature 43
3.8.3 Salinity 44
3.9 Single step growth curve 44
3.10 Protein profiles of phage UB1, phage T4 and T7 45
3.11 Extraction of phage UB1 DNA 46
3.12 Comparison of phage UB1 DNA with phage T4 and T7 47
3.13 Comparison of phage UB1 DNA with other Myoviridae phages 48
infecting E. coli O157:H7
3.14 Cloning of the phage UB1 DNA using pUC18 vector 49
3.14.1 Restriction enzyme digestion 49
3.14.2 Treatment of pUC18 vector with SAP 50
3.14.3 Ligation of DNA 50
3.14.4 Transformation of competent cells 51
3.15 Cloning of the phage UB1 DNA using CloneSmart® Blunt 52
Cloning kit (pSMART Vectors)
3.15.1 Restriction enzyme digestion 52
3.15.2 Clean up of digested DNA using GeneAll® ExpinTM 52
3.15.3 Ligation of DNA 53
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3.15.4 Transformation of competent cells 53
3.16 Purification of recombinant plasmid 54
3.17 PCR-screen of recombinant colonies 55
3.18 Sequencing of recombinant plasmid 57
3.19 Computational sequence analysis 57
3.20 PCR analysis 57
3.20.1 Primer design 57
3.20.2 Validation of PCR analysis 58

CHAPTER 4: RESULTS
4.1 Verification of phage UB1 stock 59
4.1.1 Plaques formation 59
4.1.2 Morphological study phage UB1 61
4.2 Host specificity of phage UB1 63
4.3 Viability of phage UB1 at different physicochemical attributes 64
4.3.1 pH 64
4.3.2 Temperature 66
4.3.3 Salinity 68
4.4 Single step growth curve 70
4.5 Protein profiles of phage UB1, phage T4 and T7 72
4.6 Comparison phage UB1 DNA with phage T4 and T7 74
4.7 Comparison of phage UB1 DNA with other Myoviridae phages 76
infecting E. coli O157:H7
4.8 Sequencing of phage DNA fragment by cloning using pUC18 78
vector
4.8.1 PCR-screen of recombinant colonies 78
4.8.2 Sequencing results of phage UB1 DNA fragment 80
4.8.3 Verification of sequencing results 80
4.8.4 BLASTn analysis of phage UB1 DNA fragment 82
4.9 Sequencing of phage UB1 DNA fragment by cloning using 83
CloneSmart® Blunt Cloning kits (pSMART vector)
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4.9.1 PCR-screen of recombinant colonies 83
4.9.2 Sequencing results of phage UB1 DNA fragment 85
4.9.3 BLASTn analysis of phage UB1 DNA fragment 85
4.10 Comparison of two phage UB1 DNA fragments clones using 85
pUC18 and pSMART
4.11 Comparison of morphology and restriction enzyme digestion 86
profile of phage UB1 with phage PBECO 4

CHAPTER 5: DISCUSSION
5.1 Comparative studies of phage UB1 with other coliphages 90
5.2 Potential application of phage UB1 in phage therapy 92

CHAPTER 6: RECOMMENDATIONS FOR FUTURE 96
RESEARCH

CHAPTER 7: CONCLUSION 97

REFERENCES 98

APPENDICES

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LIST OF TABLES
PAGE
Table 2.1 Overview of phage families 18
Table 3.1 Materials used and suppliers 30
Table 3.2 Stock solutions for SDS-PAGE 34
Table 3.3 Bacterial and phages strains 36
Table 3.4 Genotype of Escherichia coli strain JM109 and E. 36
cloni®
Table 3.5 Nucleotide sequences of the primers used in PCR 39
analysis
Table 3.6 Restriction enzyme digestion of phage DNA 48
Table 3.7 Restriction enzyme digestion of phage DNA and pUC18 49
vector
Table 3.8 Treatment of pUC18 vector with SAP 50
Table 3.9 Ligation of phage DNA fragment with pUC18 vector 51
Table 3.10 Ligation of phage DNA fragment with pSMART vector 53
Table 3.11 PCR-screen of recombinant pUC18 plasmid 55
Table 3.12 PCR-screen of recombinant pSMART plasmid 56
Table 3.13 PCR cycling conditions 56
Table 3.14 Nucleotide sequences of the primers used in PCR 58
analysis
Table 4.1 Host ranges of phage UB1 against selected bacteria 63
Table 4.2 Alignment result of phage DNA sequence cloned using 82
pUC18 vector

Table 4.3 Alignment result of phage DNA sequence cloned using 85
pSMART vector

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LIST OF FIGURES
PAGE
Figure 2.1 Six fundamental morphotypes of bacteriophages 7
Figure 2.2 Morphology of bacteriophage T4 9
Figure 2.3 The morphology of 13 families of phages 17
Figure 2.4 The single step growth curve of bacteriophage replication 21
Figure 2.5 The assembly of bacteriophage during lytic cycle 23
Figure 2.6 The lytic and lysogenic cycle of bacteriophage 25
Figure 3.1 Circular map and sequence reference points of the 37
pUC18 vector from SnapGene® software
Figure 3.2 Circular map and sequence reference points of the 38
pSMART® LCKan vector from Lucigen, USA
Figure 4.1 Plaques formed on bacterial lawn 60
Figure 4.2 Negatively stained electron micrograph pictures of phage 62
UB1
Figure 4.3 Effect of pH on phage viability. Data were reported as 65
mean ±SD for three replicates
Figure 4.4 Effect of temperature (⁰C) on phage viability. Data were 67
reported as mean ±SD for three replicates
Figure 4.5 Effect of salinity (NaCl % w/v) on phage stability. Data 69
were reported as mean ±SD for three replicates
Figure 4.6 Single step growth curve of phage UB1 on E. coli 71
O157:H7. Data were reported as mean ±SD for three
replicates
Figure 4.7 SDS-PAGE analysis of phage UB1, phage T4 and T7 73
structural proteins, stained with Coomasie blue staining
Figure 4.8 Comparison of the restriction enzyme digestion profile of 75
phage UB1 with phage T4 and T7

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