agbio Flashcards
1
Q
first ASEAN country initiate biotechnology regulatory system
A
Philippines
2
Q
National Committee on Biosafety Philippines (NCBP)
A
Executive No. 430 (1990)
3
Q
follow strict standards
model for member-country of ASEAN
-to become producer of agricultural biotechnology crops
A
Executive No. 430 (1990)
4
Q
recognize, one of potent tool to attain food security & sustainable agriculture
A
Agricultural Biotechnology
5
Q
ensure safety, ethical considerations, innovation
A
Biotechnology
6
Q
rapid advancement biotechnology, frameworks of laws & regulation
balance, potential benefits and potential risks
A
Biotechnology
7
Q
Address, global implication of biotechnology
A
- Cartagena Protocol on Biosafety
- Convention on Biological Diversity (CBD)
8
Q
safety transfer, handling, use of living modified orgs. (LMOs)
A
Cartagena Protocol on Biosafety
9
Q
Cartagena Protocol on Biosafety
adopted?
enter force ?
A
January 29, 2000
September 11, 2003
10
Q
- protect biological diversity from potential risks of LMOs
- advance informed agreement (AIA)
- precautionary approach
- Biosafety Clearing-House
A
Cartagena Protocol on Biosafety
11
Q
information exchange mechanism, provides info. about LMOs
A
Biosafety Clearing-House
12
Q
Biosafety Clearing-House
created, fulfill?
A
article 20 of CPB
13
Q
government designed __________
submitting information & coordination w/ BHC secretariat
A
National Focal Point (NFP)
14
Q
National Focal Point (NFP)
submitting information & coordination
A
BHC secretariat
15
Q
Department of Foreign Affairs
A
Ms. Maria Teresa T. Almojuela
16
Q
National Plant Quarantine Sevices Division
A
Mr. Ariel J. Bayot
17
Q
Department of Science and Technology (DOST)
A
Ms. Maria Lorelie U. Agbagala
18
Q
Benefits of BHC
A
Country, access to information in laws, regulation, risk
share own information in LMOs
find information, capacity building resources
19
Q
Plant Variety Protection Act of 2002
A
RA No. 9168
20
Q
National Biosafety Framework
A
EO 514
20
Q
all function pursuant to CPB
A
DA
21
Q
all function pursuant to CPB - all type of orgs.
A
NCBP
22
Q
pharmaceuticals -process foods fr. LMOs
A
DOH
23
Q
AIA, Public awareness -animal,bacteria,plant
A
DENR
24
plant genetics and crop biotechnology, prioneering
use of molecular markers
Dr. Desiree Menancio Hautea
25
ecological & environmental sciences
Dr. Flerida Carino
26
Biosafety system & Genetics
Dr. Saturnina Halos
27
BCH, biotechnology, entomology, insect resistance
Dr. Reynaldo Ebora
28
Food safety
Dr. Franco Teves
29
BCH, biotechnology, virus resistance
Dr. Dolores Ramirez
30
Mangrove forestry, plant biotechnology, forest pathology
Ms. Veronica Sinoshin
31
Biosafety system development & implementation
Biosafety legislation & regulation
Ms. Merle Palacpac
32
Biosafety system development & implementation
Biosafety legislation & regulation
Rural development
Dr. Leonardo Gonzales
33
Address issue related, conservation & sustainable use of biological diversity
Convention on Biological Diversity (CBD)
34
Specific regulation to govern biotechnology activities w/in borders
Local Regulation and Biosafety
35
Safe handling, transport, use of LMOs to minimize human & environmental risks
Biosafety
36
evaluation & approval of GMOs and biosafety products
Product approval
37
protect intellectual property right, biotech. invention
Intellectual Property Rights
38
assess environmental impact of biotechnology activity
Environmental Impact Assessment
38
Article II of 1987 Constitution
Sec 15 ,16, 17
39
Sec 15
protect & promote of right to health
40
Sec 16
balanced and healthful ecology
41
Sec 17
priority to education, science and technology
42
Article XIV 1987 Constitution
Sec 10
43
S&T, essential for nation development&progress
Article XIV 1987 Constitution
Sec 10
44
State priority to research and development,
invention, innovation, and utilization
Sec 10
44
State shall promote safe & responsible use of modern biotechnology and its product
National Policy of Biotech
45
Composition of the NCBP
1 biological scientist
1 environmental scientist
1 physical scientist
1 social scientist
2 respected members of the community
1 representative: DA, DENR, DOH
46
preventive measure, protect human health & envi.
Precautionary Principle
47
Envi. damage accountable, costs of recommendation
Polluter Pays Principle
48
Decision making, to biotechnology
Public Participation
49
Ensure, transparency & accountability to public
Transparency & Accountability
50
for developing & enforcing regulations, issuing permits
Government Agencies
51
International cooperation, technical assistance
International Organizations
52
Conduct research, provide expert advice
Scientific Institution
53
Advocate public interest, public awareness campaigns
Civil Society Organization
53
Guidance, biosafety practice & promote international cooperation in biotech application
Food and Agriculture Organization (FAO)
54
Focus, health aspect of biotech
World Health Organization
55
Consensus document, facilitate harmonization of regulatory oversight
Organization for Economic Cooperation and Development (OECD)
56
Leveraging natural processes, improve crop yield and quality
Traditional Breeding Techniques
57
enhance productivity and environmental health
Agroecology
58
use natural enemies, control pest & disease
Biocontrol Agents
59
organisms w/ altered genome, never happened in traditional reproduction or natural recombination
GMOs - Genetically modified organisms (1970s)
60
GMO start
1970s
61
Used: production
insufficient production
human insulin
bovine insulin
62
“GMO” means
change (modification)
63
occurred in the DNA, but the term doesn’t describe the change in detail.
“GMO” means a change (modification)
64
DNA from a sexually incompatible organism
“transgenic”
65
*Not all GMOs (___________), all transgenic organisms (_________)
transgenic
GMOs
66
GM but not transgenic
sexually compatible partners
67
both non-transgenic & transgenic
Genetic engineering
68
Genetic engineering
Sexual reproduction is ________
Sexual reproduction, not necessary
69
Food Quality
1.Nutritional Enhancement
2. Reduced Allergens
3. Pesticide Reduction
70
Golden Rice
higher vit & nut. content
71
Food Quantity
1. Increased Yields
2. Reduced Losses
3. Extended Shelf Life
72
Added _____% - 4 main crops
6%
(soybeans, maize, canola, cotton)
73
pollen from Bt corn
could harm monarch butterfly larvae
74
specific Bt toxin- only for animal feed but not for human consumption
StarLink Corn
75
found, taco shells & other human food products (_____)
StarLink Corn
76
elevated levels of solanine,
flawed, GM potatoes not harmful.
Poisonous Potato
77
unintended spread of genetically modified corn genes
impact on biodiversity and the integrity
Mexican Corn Gene Escape
78
StarLink Corn not approved, human consumption
2000
79
US rice contaminated w/ Bayer GMO variety
Bayer pay $750 M to 11,000 farmers
2006
80
Roundup Ready. South Korea & Japan temp. halts US wheat export
2013
81
Midwestern corn
Monsanto pays $ 250,000 to wheat grower
2014
82
Human embryonic stem cells created by somatic cell nuclear transfer
2013
82
Dolly the Sheep (1996-2003)
female Finn Dorset sheep
82
- first clone of an adult mammal
Dolly the Sheep
82
successfully cloned in 1996 by fusing the nucleus from a mammary-gland cell of a Finn Dorset ewe into an enucleated egg cell taken from a Scottish Blackface
Dolly the Sheep
82
Nuclear transfer from laboratory cells (A)
1996
83
Dolly the Sheep clone by: British biologist:
Ian Wilmut and colleagues of the Roslin Institute
83
Dolly: First mammal created by somatic cell nuclear transfer (B)
1996
83
First primate created by embryonic cell nuclear transfer (C)
1997
83
More mammals cloned by somatic cell nuclear transfer
1998-99
83
Endangered animals cloned by somatic cell nuclear transfer
2001
83
Nuclear transfer from genetically engineered laboratory cells
1997
83
technique, nucleus of a somatic (body) cell is
Somatic cell nuclear transfer (SCNT)
83
Primate embryonic stem cells created by somatic cell nuclear transfer
2007
84
allowing, begin embryonic development w/o sperm
Somatic cell nuclear transfer (SCNT)
84
DNA sequences can be_________
inserted, deleted, modified or replaced
85
broken DNA ends, and ligates
w/ little or no homology,
generating deletions or insertions
Non-homologous end joining (NHEJ)
86
Use undamaged DNA template, homologous
chromosome to repair break
Homology-directed repair (HDR)
87
introduce cuts to the DNA
Nucleases
88
Type of nuclease
1. meganucleases
2. zinc finger nucleases,
3. TALEN
4. CRISPR/Cas9
89
engineered nucleases for precise genome editing
TALEN
Transcription Activator-Like Effector-based Nucleases
90
customizable & bind to any specific DNA seq.
TALE DNA-Binding Domain
91
cuts, DNA guided by the TALE domain.
requires dimerization to function,
meaning two TALEN modules must target adjacent DNA sequences to create a double-strand break
FokI Nuclease Domain
92
clustered regularly interspaced palindromic repeats
CRISPR/Cas System
93
CRISPR
associated endonuclease
94
first Cas used
Cas9
95
most popular genome editing methods
CRISPR/Cas System
96
Components
sgRNA
Cas endonuclease
96
Stages
(1) DNA damage generation such as double strand breaks
(2) host cell repair of damaged sites
97
makes specific protein-DNA binding
TALEN
98
makes specific RNA-DNA binding
CRISPR
99
guide RNA that locates the correct segment of host DNA along with a region that binds to tracrRNA (generally in a hairpin loop form), forming an active complex
crRNA
100
Binds to crRNA and forms an active complex
tracrRNA
101
RNA consisting of a tracrRNA & at least one crRNA
sgRNA
102
active form, to modify DNA (encodes proteins that unwind and cut the DNA)
Cas9
103
template during the host cell's DNA repair process, allowing for the insertion of a specific DNA sequence at the site broken by Cas9.
Repair template
104
more precision than Cas9 is _____
Cas12a
105
Cas12- not need
tracrRNA
106
Cas 12a makes
staggered cut
107
Cas9 -makes
blunt end cut
108
109
CRISPR-induced double-strand break can induce gene “knock-out” by utilizing the NHEJ, and as an error-prone, it results in genomic deletions or insertions
Gene Silencing or “Knock-Out”
109
for DNA-free gene editing w/o DNA vectors, requiring only RNA or protein components.
good choice to avoid the possibility of undesirable genetic alterations
DNA-Free Gene Editing
109
through HDR as a precise insertion of a donor template
Gene Insertions or “Knock-ins”
109
modifying the Cas9 protein so it cannot cut DNA. modified Cas9, led by a sgRNA, targets the promoter region of a gene and reduces transcriptional activity and gene expression.
Transient Gene Silencing
109
determines the order of the four chemical building blocks - called "bases" - that make up the DNA molecule.
DNA sequencing
110
sequence tells scientists the kind of genetic information that is carried in a particular DNA segment
DNA double helix
110
four chemical bases (A, T, C, and G) *base pair
DNA double helix
110
Maxam-Gilbert
STEPS:
1. DNA sample
2. Preparation of DNA into single strand
3. Amplification
4. Addition of P32 as 5’ phosphate
5. Cleave a specific nucleotide by using chemical
6. Load DNA fragment into gel and analyze
110
- radioactive labeling 5′-P ends of double-stranded DNA
phosphate group (P32) using polynucleotide kinase
Maxam-Gilbert
110
- DNA, denatured w/ DiMethyl SulfOxide (DMSO) 90°C
- the resulting ssDNA molecules are segregated via
Electrophoresis
Maxam-Gilbert
111
Not routinely, disadvantages such as:
- high toxicity, phosphate isotope & cutting chemical
usage
- difficulties analyzing sequences longer than 500bp
- errors during cleavage
112
-based on chain elongation termination, use in DNA seq.
w/ help of _________& ___________
Sanger Method (1977)
polymerase and special nucleotides
113
Sanger Sequencing
reagent
1. DNA sample
2. Preparation of DNA into single strand
3. Amplification
4. Sanger sequencing
5. Fluorescently labelled DNA sample
6. Capillary gel electrophoresis and analysis of the complementary sequence
114
for identifying genes
DNA sequence
115
now use in cattle farming & other animals
precision selective breeding methods
Genome Sequencing
116
combination of DNAs of different origins
Recombinant DNA molecule
117
combination possible by the use of
restriction enzymes and ligase
118
Recombinant DNA molecule
technique called
molecular or DNA cloning
118
* introduced into a host cell as bacterium
* bacterium divides and forms a colony,
each cell in the colony contains one or more identical copies
of the recombinant DNA.
Recombinant DNA molecule
119
Two categories of enzymes needed
o restriction endonucleases
o DNA ligase
120
- scissors, cut both strand of DNA sugar-phosphate backbone
o restriction endonucleases
121
– joins two DNA molecules
o DNA ligase
122
found primarily in bacteria
o restriction endonucleases
123
both strands, equal length, have no unpaired bases
Blunt end
124
one strand, longer than the other, has unpaired bases
Sticky end
125
can form hydrogen bonds with complementary bases
helpful in cloning, can be joined by ________
Sticky end
DNA ligase
126
* Restriction endonucleases
recognize specific sequences
4-6 base pairs (bp) and palindromic.
cuts particular place within that sequence.
127
enzyme makes the cut by ________
breaking covalent bonds
128
* Restriction enzyme
- blunt ends / staggered ends (or cohesive or sticky ends)
129
* DNA ligase
- sticky ends or blunt ends
130
o first letter –
o next two letters –
o R-
o I -
– first letter of genus name
– first two letters of species name
for strain type
for the first enzyme of that type
131
1,000 bp away from recognition site.
Type I
132
cut directly within recognition site.
Type II
133
25 bp of the recognition site.
Type III
134
*EcoB and EcoK
type I enzymes
135
*Hind II and Hind III
type II enzymes
136
catalyzes formation of covalent bonds
Ligase
137
- does not discriminate DNA w/ different origins
Ligase
138
DNA ligase joins two different DNA fragments
one DNA molecule, a recombinant DNA.
139
transfer to host cell, replicated (amplified)
recombinant DNA
140
*transfer use, suitable DNA delivery method:
electroporation
141
set of experimental techniques to generate a population of
organisms, same molecule of recombinant DNA
DNA cloning (a.k.a. Molecular cloning)
142
formed in vitro by inserting DNA fragments of interest into vector DNA molecules.
Recombinant DNA molecules
143
Specific Steps in Cloning
depends on
type of DNA used, host cell types, a goal of cloning
144
CLONING
Broad steps:
1. Isolation of DNA vector and target gene
2. Cutting both DNAs using the restriction enzymes
3. Ligating the target gene into vector
4. Transformation of host cell with the recombinant DNA
5. Cloning and selecting cells harboring the recombinant
DNA
145
- DNA molecules, used as vehicles in transporting foreign piece of DNA into host cell
Cloning vectors
146
Cloning vectors *available for
bacteria, yeast, fungi, animals and plants
147
replicate independently in a host cell
Cloning vectors
147
Important characteristics of cloning vectors
origin of replication
Small enough to be isolated
several unique restriction sites
selectable markers, for identifying
147
- small, double strands of bacterial DNA, extrachromosomal
Plasmid Vectors
148
- DNA fragments up to 10 kb (kilobases)
Plasmid Vectors
149
- transported into bacterial cells
- digestion, ligation and transformation are inefficient
Plasmid Vectors
150
white bacterial colonies- gene of interest (recombinants)
blue- do not have the gene of interest (nonrecombinants)
Blue-White screening
151
gene of interest (recombinants)
white bacterial colonies
151
do not have the gene of interest (nonrecombinants)
blue
152
- Viral DNA can engineered for use as a cloning vector
Phage DNA Vectors
153
- most abundant biological agent
Phage DNA Vectors
153
Bacteriophages, also known as ______
phages
154
Phage DNA Vectors_________ kb λ DNA
50 kb λ DNA
154
viruses quickly take over the host cell, make many copies, break the cell, and infect other cells.
lytic cycle
155
viruses sneak into the host's DNA, stay hidden, and wait
lysogenic cycle
156
packaged into a protein coat in vitro and made to infect bacterial cells
Cosmid Vectors
157
recombinant cosmid replicates as a plasmid and the host cells are not lysed
Cosmid Vectors
158
Cosmid Vectors _________kb
35 to 45 kb
159
useful for analyzing large portions of complex genomes
Bacterial Artificial Chromosomes (BAC)
159
widely used in large genome sequencing projects
Bacterial Artificial Chromosomes (BAC)
159
Bacterial Artificial Chromosomes (BAC) _________ kb of DNA.
100 to 300 kb of DNA.
160
lacks some genes that cause
tumor
160
Yeast Artificial Chromosomes (YAC) _____kb
200 and 1500 kb
160
useful for eukaryotic molecular studies
Yeast Artificial Chromosomes (YAC)
160
special region in the Ti plasmid, called T-DNA
Plant Cloning Vector
161
lacks some genes that cause tumor
Plant Cloning Vector
162
to transfer foreign genes into plants
T-DNA
163
inserted into the T-DNA region
Foreign DNA
164
DNA Cloning Review & Applications
1. Cutting and pasting DNA
2. Bacterial transformation and selection
3. Protein production
165
Genetic code deciphered
1965
166
Recombinant DNA method
1973
167
Chemically synthesized DNA active in cells
1976
168
Genentech founded
1976
169
Human insulin produced in E. coli
1979
169
Human somatostatin produced in E. coli
1977
170
Humulin approved by FDA
1982
171
process, foreign DNA transferred to host cells for applications such as genetic research or gene therapy.
Gene delivery
172
utilize bacterial or viral carriers
Biological or Indirect
173
include certain polymers and lipids
chemical methods
174
utilize physical properties and forces to transport genetic material into cells.
physical methods
175
approaches to deliver GE reagents
mostly rely on Agrobacterium-mediated transformation or particle bombardment (aka gene gun or biolistic method)
Traditional approaches
176
177
178
179
180
181
182
gene delivery via a virus.
transduction
182
insertion of exogenous DNA into prokaryotic cell
transformation
182
Advantage of gene delivery
● Viruses, high transduction efficiencies yield up to 100% transduction efficiency in vitro.
● Viruses, naturally introduce genetic material into cells.
182
gene delivery via nonviral
transfection
183
In plants
Geminiviruses
183
delivers, RNA into cells
184
Retroviruses _________bases
8000 bases of ssRNA
185
can yield permanent transduction
(delivered genes, remainder of its life)
Retroviruses
186
nonenveloped viruses that carry linear, dsDNA
Adenoviruses
186
infecting mucosal linings
Adenoviruses
186
Geminiviruses
family ?
186
ability to transfer T-DNA in its Ti plasmid into plant cells
Agrobacterium tumefaciens bacterium
186
process of using Agrobacterium to transfer a gene of interest into the plant cells
Agrobacterium-mediated
186
ability to infect a wide variety of plant species
like wheat, maize, cotton, and more.
Geminiviruses
187
a soil pathogenic bacterium; causes crown-gall disease or hairy root disease in infected dicotyledonous plants.
Agrobacterium
187
By replacing its ________with desired gene/s, Agrobacterium is able to transfer the desired genes into the genome of target plants.
T-DNA
188
Three main components prepare Agrobacterium-mediated transformation:
1. T-binary system
2. Agrobacterium Competent Cells
3. Plant
189
at top as a region containing (MCS)
T-DNA
190
to insert a gene of interest
MCS or multiple cloning site
191
the plant selectable marker and bacterial marker
Components for selection
191
origin of replication for Agrobacterium (OriA) and origin of replication for Escherichia coli (OriE).
Components for replication
191
monitoring the recombinant protein in the transformed plant
a reporter
191
Components for gene expression these elements include promoter, ________
polyA signals
192
are cells able to take up the T-binary vector containing T-DNA and the gene of interest
Agrobacterium Competent Cells
193
The transformation of plant cells by using
Agrobacterium commonly involves
incubating the cells or tissues with the bacteria.
194
Microparticles on the order of ~0.5-1μm are used, typically made of gold. (Gold can ionize positively, interacting well with the negatively charged DNA or RNA.)
1. Gene Gun
(biolistic delivery system/ particle bombardment)
195
Involves using extremely fine needles to penetrate the cell’s plasma membrane; creates a small hole in the membrane to deliver material inside.
Microinjection
196
Microinjection
Advantage:
If done correctly, the membrane reseals after needle removal, causing minimal cellular damage.
197
Typical polymers that can be used for gene delivery
include, but certainly are not limited to, poly(l-lysine) (PLL), poly(ethylenimine) (PEI), and chitosan.
* Polymers
197
very stressful to the cell
Electroporation
198
used for gene delivery in a process termed lipofection.
* Lipids
199
Because of the aqueous environment inside cells and tissues, the hydrophobic tails of the lipids will ___________, the interiors of
which can contain ________ for cellular
delivery.
coalesce to form hollow liposomes
oligonucleotides
200
PEG
mediated Transformation
201
- facilitates the uptake of foreign
DNA into the protoplasts by inducing transient pore formation
in the plasma membrane, allowing the DNA to enter the cells.
PEG (Polyethylene Glycol)
202
plant cells w/ cell walls removed, leaving only the plasma membrane intact. Protoplasts are used as the target cells for introducing foreign DNA, as their lack of cell walls makes it easier for the DNA to enter the cell.
Protoplasts
203
acts as osmotic stabilizer to help maintain the integrity and viability of the protoplasts
Mannitol
204
Direct Delivery
1. Particle delivery method
2. Electroporation
3. Agrobacterium-mediated method
205
Particle delivery method
* RR/ Roundup Ready® crops
206
Electroporation
* InVigor™ Maize (Glufosinate herbicide tolerance, PC)
* Biological/ Indirect Delivery
207
Agrobacterium-mediated method
* Bt crops
* Golden/ Malusog Rice
208
widely used in gene transfer
reporter gene
Selectable markers
209
gene, attached to the regulatory sequence of another gene of interest
reporter gene
210
artificial selection of transformed cells; usually they are antibiotic or herbicide resistance genes
Selectable markers
211
kanamycin resistance confer selective advantage to the host so that cells that have it survive in a selection medium containing the antibiotic
Positive selection markers
212
the opposite is true
negative selection markers
213
remove unwanted genes from the plant's genome, ensuring that the final plant does not carry any marker genes.
Gene Deletion Technologies
214
scientists introduce two separate sets of genes into the plant cells: one for the gene of interest and one for the marker gene.
Co-Transformation:
215
palatinose system involves using a gene that makes the plant survive in the presence of palatinose, a sugar. Only the transformed plants survive because they can process the sugar, while the untransformed plants die. This eliminates the need for antibiotic resistance genes altogether
Safer Marker Systems
216
antibiotic selection marker genes lead to _______
lead to antibiotic resistance
217
origin of replication for Agrobacterium (_______) and origin
of replication for Escherichia coli (_______).
OriA
OriE)
218
Impacts on Health and Medicine
Potential Risks
1. Allergic Risk
2. Toxicity
3. Antibiotic Resistance
219
Impacts on the Environment
Benefits
1. Reduced Pesticide
2. Improved Weed Control
3. Environmental Benefits
220
Impacts on the Environment
Potential Risks
1. Cross-Pollination
2. Herbicide-Resistant Weeds
3. Pesticide Adaptation
220
D. Impacts on the Economy
Benefits
GM technology- positive impact
2012 $18.8 billion
Added 6% - 4 main crops
(soybeans, maize, canola, cotton)
221
D. Impacts on the Economy
Potential Risks
1. Higher Seed Costs
2. Trade Barriers
3. Corporate Dependency
4. Small-Scale Farmer Impact
