Biotechnology: Principles, Applications, And Social Implications

gooseliverBiotechnology

Oct 22, 2013 (3 years and 7 months ago)

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Biotechnology:

Principles, Applications,

and Social Implications


From Protein to Product

Phil McClean

Department of Plant Science

North Dakota State University

The techniques used by the biotechnology industry

to modify genes and introduce them into transgenic organisms


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What is Biotechnology
?

How about some definitions

General Definition

The application of technology to improve


a biological organism


Detailed Definition

The application of the technology to modify the

biological function of an organism by adding genes

from another organism

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But we know nature
does not

have

all of the traits we need



Here we see bean has
many



seedcoat colors and patterns


in nature


Nature has a
rich source of variation


These definitions imply biotechnology

is needed because:

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But nature
does not

contain all the

genetic

variation

man desires


Fruits with vaccines


Grains with improved nutrition

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What controls this natural variation?

Allelic

differences at
genes

control a specific trait

Gene
-

a piece of DNA that controls the


expression of a trait


Allele
-

the alternate forms of a gene

Definitions are needed for this statement:

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What is the difference between

genes and alleles for Mendel’s Traits?

Mendel’s Genes

Plant height

Seed shape

Tall

Short

Allele

Smooth

Wrinkled

Allele


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This Implies a

Genetic Continuum

A
direct relationship

exists between the gene, its alleles,

and the phenotypes (different forms ) of the trait

Alleles must be:



similar

enough to control the same trait



but
different

enough to create different phenotypes

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Allelic Differences for Mendel’s Genes

Plant Height Gene

Gene:
gibberellin 3
-

-
hydroxylase

Function:
adds hydoxyl group to GA
20

to make GA
1

Role of GA
1
:
regulates cell division and elongation

Mutation in short allele:
a single nucleotide converts


an alanine to threonine in final protein

Effect of mutation:
mutant protein is 1/20 as active

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Gene:
strach branching enzyme (SBE) isoform 1

Function:
adds branch chains to starch

Mutation in short allele:
transposon insertion

Effect of mutation:
no SBE activity; less starch, more


sucrose, more water; during maturation seed looses


more water and wrinkles

Allelic Differences for Mendel’s

Seed Shape Gene

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Central Dogma of Molecular Genetics

(The guiding principle that controls trait expression)

DNA

(gene)

RNA

Protein

Trait

(or phenotype)

Transcription

Translation

Plant height

Seed shape

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In General, Plant Biotechnology Techniques

Fall Into Two Classes



Identify a gene from
another species

which controls


a trait of interest



Or modify an existing gene (create a new allele)

Gene Manipulation



Introduces that gene into an organism



Technique called
transformation



Forms
transgenic organisms

Gene Introduction

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Gene Manipulation Starts

At the DNA Level

The nucleus

contains DNA

Source
: Access Excellence

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DNA Is Packaged

Source
: Access Excellence

Double
-
stranded

DNA

Chromosomes

is condensed

into

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Chromosomes Contain Genes

Chromosome

Gene

Source
: Access Excellence

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Genes Are Cloned Based On:

Similarity to known genes

Homology cloning

(mouse clone used to obtain human gene)

Protein sequence

Complementary genetics

(predicting gene sequence


from protein)

Chromosomal location

Map
-
based cloning

(using genetic approach)

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Human clone

library

Clones transferred

to filter

Mouse probe

added to filter

Hot
-
spots are human

homologs

to mouse gene

Homology Cloning

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Complementary Genetics

1. Protein sequence is related to gene sequence

NH
3
+
-
Met
-
Asp
-
Gly
--------------
Trp
-
Ser
-
Lys
-
COO
-


ATG GAT
-
GCT TGG
-
AGT
-
AAA


C C C G


A TCT


G C


A


G

2. The genetic code information is used to design PCR primers

Forward primer:
5’
-
ATGGAT/CGCN
-
3’

Reverse primer:
5’
-
T/CTTNC/GT/ACCA
-
3’

Notes:
T/C = a mixture of T and C at this position;


N = a mixture of all four nucleotides


Reverse primer is the reverse complement of the gene sequence

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3. Use PCR to amplify gene fragment

Complementary Genetics

(cont.)

a. template DNA is melted (94C)

3’ 5’

5’ 3’

3’ 5’


5’ 3’

b. primers anneal to complementary site in melted DNA (55C)

3’ 5’


5’ 3’

3’ 5’


5’ 3’

c. two copies of the template DNA made (72C)

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Denaturation:
DNA melts

Annealing:
Primers bind

Extension:
DNA is replicated

PCR Animation

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PCR Again

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Human clone

library

Clones transferred

to filter

PCR fragment

probe added to filter

Hot
-
spots are
human gene

of interest

Complementary Genetics

(cont.)

4. Gene fragment used to screen library

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Map
-
based Cloning

1. Use genetic techniques to

find marker near gene

Gene

Marker

2. Find cosegregating marker

Gene/Marker

3. Discover overlapping clones


(or contig) that contains the marker

Gene/Marker

4. Find ORFs on contig

Gene/Marker

5. Prove one ORF is the gene by


transformation or mutant analysis

Mutant +
ORF

= Wild type?

Yes?
ORF

= Gene

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Gene Manipulation



It is now routine to isolate genes



But the target gene must be carefully chosen



Target gene is chosen based on desired phenotype

Function:

Glyphosate (RoundUp) resistance


EPSP synthase enzyme

Increased Vitamin A content


Vitamin A biosynthetic pathway enzymes

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The RoundUp Ready Story



Glyphosate is a broad
-
spectrum herbicide



Active ingredient in RoundUp herbicide



Kills all plants it come in contact with



Inhibits a key enzyme (
EPSP synthase
) in an amino acid pathway



Plants die because they lack the key amino acids



A resistant EPSP synthase gene allows crops


to survive spraying

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+ Glyphosate

X

RoundUp Sensitive Plants

X

X

Shikimic acid + Phosphoenol pyruvate

3
-
Enolpyruvyl shikimic acid
-
5
-
phosphate

(EPSP)

Plant

EPSP synthase

Aromatic

amino acids

Without amino acids,
plant dies

X

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Bacterial

EPSP synthase

Shikimic acid + Phosphoenol pyruvate

3
-
enolpyruvyl shikimic acid
-
5
-
phosphate

(EPSP)

Aromatic

amino acids

RoundUp Resistant Plants

+ Glyphosate

With amino acids,
plant lives

RoundUp has no effect;

enzyme is resistant to herbicide

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The Golden Rice Story



Vitamin A deficiency is a major health problem



Causes blindness



Influences severity of diarrhea, measles



>100 million children suffer from the problem



For many countries, the infrastructure doesn’t exist

to deliver vitamin pills



Improved vitamin A content in widely consumed crops

an attractive alternative

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-
Carotene Pathway in Plants

IPP

Geranylgeranyl diphosphate

Phytoene

Lycopene



-
捡rot敮e

(v楴im楮iA⁰r散畲獯爩

Phytoene synthase

Phytoene desaturase

Lycopene
-
beta
-
cyclase

ξ
-
carotene desaturase


Problem:

Rice lacks

these enzymes

Normal

Vitamin A

“Deficient”

Rice

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The Golden Rice Solution

IPP

Geranylgeranyl diphosphate

Phytoene

Lycopene



-
捡rot敮e

(v楴im楮iA⁰r散畲獯爩

Phytoene synthase

Phytoene desaturase

Lycopene
-
beta
-
cyclase

ξ
-
carotene desaturase


Daffodil gene

Single bacterial gene;

performs both functions

Daffodil gene


-
Carotene Pathway Genes Added

Vitamin A

Pathway

is complete

and functional

Golden

Rice

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Metabolic Pathways are Complex

and Interrelated

Understanding pathways
is
critical

to developing
new products

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Modifying Pathway Components

Can Produce New Products

Modified Lipids =

New Industrial Oils

Turn On Vitamin Genes =
Relieve Deficiency

Increase amino acids =

Improved Nutrition

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Trait/Gene Examples

RoundUp Ready

Bacterial EPSP

Golden Rice

Complete Pathway

Plant Virus Resistance

Viral Coat Protein

Male Sterility

Barnase

Plant Bacterial Resistance

p35

Salt tolerance

AtNHX1

Trait

Gene

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Introducing the Gene or

Developing Transgenics

Steps

1. Create transformation cassette

2. Introduce and select for transformants

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Transformation Cassettes

Contains

1. Gene of interest



The coding region and its controlling elements

2. Selectable marker



Distinguishes transformed/untransformed plants

3. Insertion sequences



Aids
Agrobacterium

insertion

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Gene of Interest

Coding Region



Encodes protein product

ex.: EPSP



-
捡c潴敮攠来湥g

Promoter Region



Controls when, where and how much the gene is expressed

ex.: CaMV35S (constitutive; on always)


Glutelin 1 (only in rice endosperm during seed development)

Promoter

Coding Region

TP

Transit Peptide



Targets protein to correct organelle

ex.: RbCS (RUBISCO small subunit; choloroplast target

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Selectable Marker

Coding Region



Gene that breaks down a toxic compound;

non
-
transgenic plants die

ex.:
nptII

[kanamycin (bacterial antibiotic) resistance]


aphIV

[hygromycin (bacterial antibiotic) resistance]


Bar

[glufosinate (herbicide) resistance]

Promoter Region



Normally constitutive

ex.: CaMV35s (Cauliflower Mosaic Virus 35S RNA promoter

Promoter

Coding Region

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Effect of Selectable Marker

Transgenic =
Has Kan or Bar Gene

Plant grows in presence

of selective compound

Plant dies in presence

of selective compound

Non
-
transgenic =
Lacks Kan or Bar Gene

X

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Insertion Sequences



Used for Agrobacterium
-
transformation

ex.: Right and Left borders of T
-
DNA

Required for proper gene insertions

T
L

T
R

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Let’s Build A Complex Cassette

pB19hpc (Golden Rice Cassette)

T
L

T
R

aphIV

35S

Gt1

psy

35S

rbcS

crtl

Hygromycin

Resistance

Phytoene

Synthase

Phytoene

Desaturase

T
-
DNA

Border

T
-
DNA

Border

Selectable

Marker

Gene of

Interest

Gene of

Interest

Insertion

Sequence

Insertion

Sequence

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Transformation cassettes are developed in the lab



They are then introduced into a plant



Two major delivery methods

Delivering the Gene

to the Plant



Agrobacterium



Gene Gun

Tissue culture

required to generate

transgenic plants

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Plant Tissue Culture

A Requirement for Transgenic Development

A plant part

Is cultured

Callus

grows

Shoots

develop

Shoots are rooted;

plant grows to maturity

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Agrobacterium

A natural DNA delivery system



A plant pathogen found in nature



Hormone genes expressed and galls form at infection site



Delivers DNA that encodes for plant hormones



Infects many plant species

Gall on

stem

Gall on

leaf



DNA
incorporates

into plant chromosome

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The Galls Can Be Huge

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Natural Infection Process Is Complex

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But Nature’s
Agrobacterium

Has Problems

Infected tissues cannot be regenerated (via tissue culture)

into new plants

Transferred DNA (T
-
DNA) modified by



Removing phytohormone genes



Retaining essential transfer sequences



Adding cloning site for gene of interest



Phytohormone balance incorrect regeneration

Solution?

Why?

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The Gene Gun



DNA vector is coated onto gold or tungsten particles



Particles are accelerated at high speeds by the gun



Particles enter plant tissue



DNA enters the nucleus and


incorporates into chromosome



Integration process unknown

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Transformation Steps

Prepare tissue for transformation

Introduce DNA

Culture plant tissue



Develop shoots



Root the shoots

Field test the plants



Leaf, germinating seed, immature embryos



Tissue must be capable of developing into normal plants



Agrobacterium

or gene gun



Multiple sites, multiple years

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The Lab Steps

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Lab Testing The Transgenics

Insect Resistance

Transgene=

Bt
-
toxin protein

Cold Tolerance

Transgene=

CBF transcription factors

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Salt Tolerant

Mercury Resistance

More Modern Examples

Transgene=

Glyoxylase I

Transgene=

Mercuric ion reductase

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The Next Test Is The Field

Non
-
transgenics

Transgenics

Herbicide Resistance

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Final Test

Consumer Acceptance

RoundUp Ready Corn

Before

After

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The Public Controversy



Should we develop transgenics?



Should we release transgenics?



Are transgenics safe?



Are transgenics a threat to non
-
transgenic


production systems?



Are transgenics a threat to natural


eco
-
systems?