CONTENTS

Topics Page No.

Acknowledgement I
Abstract II
Table of Contents III
List of Tables VI
List of Figures VII
Abbreviations IX

CHAPTER ONE : Introduction 1-6

CHAPTER TWO : Vectors for improvement of crops 7-130

2.1 Plasmid 9
2.1.1 Types of Plasmid 12
2.1.2 Plasmid DNA extraction 13
2.1.3 Conformations 13
2.1.4 pBR322 vector 15
2.1.5 pUC vector 18
2.1.5.1 pUC18/19 22
2.1.5.2 pUC57 23
2.1.5.3 pUC 118 and 119 24
2.1.6 Phagemids 26
2.1.7 The pET vector system 27
2.1.8 The pTrcHis vector system 30
2.1.9 Ti plasmid 32
2.1.9.1 Agrobacterium tumefaciens T-DNA transfer process 33
2.1.9.1.1 Agobacterium and invention of golden rice 43
2.1.9.2 Strtegies for inserting foreign gene into Ti plasmid 48
Binary Vectors 49
Basic Structure of Binary Vectors 50
Binary vector type 55
Superbinary vector 58
Cointegrate vector 64

III
 2.1.10 The Ri plasmid 66
2.2 Yeast plasmid vector 67
2.2.1 YIp vectors 70
2. 2. 2 YEp vectors 71
2.2.3 YCp vectors 74
2.2.4 Yeast replicative plasmids (YRps) 74
2.3 Yeast artificial chromosme 76
2.3.1 Advantage of YAC 77
2.3.2 Features of YACs 78
2.4 Bacterial artificial chromosome 83
2.4.1 Application of BACs 84
2.4.2 Construction of BACs 87
2.5 Bacteriophage 102
2.5.1 Double-stranded phage vectors 102
2.5.2 Single stranded or filamentous phages 103
2.5.2.1 M13 104
2.5.2.2 Lambda phage 105
2.5.2.2.1 Development of lambda: Two alternative modes 106
2.5.2.2.2 Lamda as cloning vector (λ) 106
Novel Lambda Vectors 112
Screening a Phage Library 118
7.5.3 Caulimovirus vectors 122
7.5.4 Geminivirus vectors 122
2.6 Cosmid 123
2.6.1 Cloning into Cosmid Vector 124
2.6.2 Problems associated with lambda and cosmid cloning 125
2.6.3Advantages of Cosmid cloning vector 126
2.7 Transposon 126

CHAPTER THREE: Application 131-142

3.1 Agricultural applications

3.2 Food application 135
3.3 In health sector 136

IV
CHAPTER FOUR: Issues Regarding the Use of Cloning Vectors for
GM Crop Development
143-148

4.1 The concerns about GM food 145

4.2 Antibiotics 145
4.3 Threats to wildlife 145
4.4 Contamination 146
4.5 Liability 147
4.6 The impact of European and international trade policy 148

CHAPTER FIVE : Discussion 149-153

GLOSSARY 154-163

REFERENCES 164-188

V
 LIST OF TABLES
Title of Tables Page No
Table 2.1: The size of the insertion gene and its probability of transfer 10
Table2.2: Features of the pET System Vectors 28
Table 2.3: Well-known binary and superbinary vectors 63
Table 2.4: Components of common yeast plasmid vectors 69
Table 2. .5: Plant BAC libraries constructed by different scientist. 87
Table 2.6: Cloning sites of insertion vectors 108
Table 2.7: Positions of restriction sites and lengths of arms and stuffers in 112
replacement vectors.
Table 2.8. Novel lambda vectors 113
Table 3.1: Antigens produced in transgenic plants 138
Table 3.2: Transient production of antigens in plants after infection with plant 140
viruses expressing a recombinant gene
Table 3.3: Antibodies and antibody fragments produced in transgenic plants 141

LIST OF FIGURES

Title of Figures Page

No.

VI
Figure: 2.1: Cloning into a plasmid 11
Figure 2.2: pBR322 plasmid 15
Fig 2.3: Determination of the transformants by the color of the colonies 16
Fig 2.4:Selection of positive clones by replica plating 17
Figure 2.5: pUC vector 18
Figure 2.6: pUC polylinker region 21
Figure 2.7:pUC18/19 vector 22
Figure 2.8:pUC57 vector 24
Fig 2.9: pUC 118/119 26
Figure 2.10: pET vector 27
Figure 2.11:pET reading frame 29
Figure 2.12:pTrcHis vector 30
Figure 2.13: EK cleavage site 32
Figure 2.14:T-DNA borders 33
Figure 2.15:T-DNA transfer into plant genome 34
Fig 2.16: Prevalence of vitamin A deficiency 44
Fig 2.17: White rice and golden rice 44
Fig 2.18: A simplified overview of the carotenoid biosynthesis pathway in 46
golden rice
Fig 2.19: Gene construct used to generate Golden Rice. 46
Fig 2.20: Progress made since the proof-of-concept stage of Golden Rice 48
Figure 2.21: Typical structure of a binary vector. Key components and their 51
major options are displayed
Figure 2.22:Final step of construction of a superbinary vector 62
Figure 2.23:A typical cointegrate vector 65
Figure 2.24:The cointegration strategy 66
Figure 2.25:Yip5 vector 70
Figure 2.26: Two-step gene replacement 71
Figure 2.27:YEp Yeast episomal plasmid recombination 72
Figure 2.28: The genome of a diploid cell of S. cerevisiae 73
Figure 2.29: YRp vector 75
Figure 2.30: diagram of a pYAC4 80
Fig 2.31: A typical overall scheme of the YAC cloning system 81
Figure 2.32: pBeloBAC11 86
Figure 2.333:cutting of dephosphorilated vector 89

VII
Figure 2.34: Diagram of a mini-gel 89
Figure 2.35: The components of the Gibco BRL Cell-Porator. 91
Figure 2.36: Diagram of BACs digested with NotI 92
Figure 2.37: CHEF analysis of DNA from plugs of grape 94
Figure 2.38: Diagram of a typical test digest of high MW DNA 95
Figure 2.39: Performing the first size selection 97
Figure 2.40: The second size selection. 100
Figure 2.41: Structure of M13 104
Figure 2.42: Regulatory region of the lambda genome. Binding sites of specific 105
gene products are indicated by the arrowheads.
Figure 2.43: Use of phage lambda as a DNA cloning vehicle 111
Figure 2.44: Cloning by using cosmid vectors 125
Fig 3.1: Strategies for expression of antigen in plant 139

ABBREVIATIONS

VIII
Ac Activator
BHR Broad host range
CHEF Contour-clamped homogeneous electric field
Ds Dissociation
EK Enterokinase
FISH Fluorescence in situ hybridization
GM Genetically modified
LB Left border
LMP Low Melting Point
LPS Lipopolysaccharides
MCS Multiple cloning site
NLS Nuclear location signals
PCR- Polymerase chain reaction
PFGE Pulsed-field gel electrophoresis
PSK Postsegregational killing system
RB Right border
SCIMAC Supply Chain Initiative on Modified Agricultural Crops
SS Single-stranded
Ti plasmid- Tumor inducing plasmid
TM- Transmembrane
TrC Trailer complementary

IX