Bcmb 612

2018/2019

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 Course overview
• Objectives:
introduce students to applications of
biotechnology (specifically health, energy,
industrial, environmental and agricultural
applications).

Highlight basic biochemical principles behind

these applications and challenge students to think
about potential applications and research
opportunities within the local environment. 2
 My topics
• Agriculture:
• Plant agriculture; genetic engineering in plants; GM
food crops; cell and tissue culture; biological
fertilizers;
• Animal Agriculture; Veterinary medicine;
reproductive manipulation; Molecular farming.

• Human health:
• Forensics and genealogy;
• Disease; types, diagnostic methods and treatment of
disease; 3
 History: Early biotechnology
• One of the earliest examples is the use of yeast to
produce beer and bread.

• And early in the 20th century, researchers

discovered a mold that was used to create
penicillin
• one of the most widely used antibiotics in the world

• Biotechnology is not only about complex machine

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 History of genetic engineering
• Humans have domesticated plants and animals since
around 12,000 BCE, using selective breeding or artificial
selection

• In selective breeding
– organisms with desired traits (and thus with the desired genes)
are used to breed the next generation
– organisms lacking the trait are not bred,

• Selective breeding is a precursor to the modern concept of

genetic modification
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 Plant breeding

plant breeding is the process of using two parent plants to create an “offspring” plant.
Which will share characteristics of each of its parents, so a new seed will share
characteristics of the “mother” and “father” plants that created it.
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Ancient to present

For more than 10,000 years,

mankind has selectively
bred plants and animals.

The cows you see in

farmer’s fields bear little
resemblance to the ancient
Aurochs from which they
descended.

And the corn you eat is the

domesticated version of a
wild grass called teosinte.

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 History of GMO/ recombinant DNA
In 1972 Paul Berg created the first recombinant DNA molecule when he combined
DNA from a monkey virus with that of the lambda virus

1973
Bacterial gene KanR cloning
into another
bacteria
KanR

transformation
Herbert Boyer and Stanley
Cohen created the first
Bacteria now resistant to Kan GMO in 1973

Boyer and Cohen expressed other genes in bacteria.

in 1974 they put genes from a toad (Xenopus laevis) into bacteria, creating the first
GMO expressing a gene from an organism from different kingdom

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https://en.wikipedia.org/wiki/Genetically_modified_organism
 What are GMOs?
• any organism in which the genes or other
genetic material have been modified by using
in vitro (recombinant DNA) techniques.
– A plant, such as corn or soybean, is considered
genetically modified when genetic material from
outside of that organism is inserted into its DNA
sequence.
– Plants grown from seed harvested from
genetically modified plants will also contain the
genetic modification.
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 GMO
• A genetically modified organism (GMO) is any organism
whose genetic material has been altered using genetic
engineering techniques (i.e., a genetically engineered
organism).

• GMO is a "transgenic organism."

– This is an organism whose genetic makeup has been altered by
the addition of genetic material from an unrelated organism.

– The first genetically modified mouse was in 1973, and the first
plant was produced in 1983

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 Applying science to breeding

Marker genes which

include
disease resistance,
drought tolerance,
yield,
taste,
nutrition,

In the past, breeders crossed and planted to determine progeny identity

Now markers are identified, inserted and then identified in progeny (rapid and high tech)
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http://www.monsanto.com/improvingagriculture/pages/modern-breeding-techniques.aspx
 How to make GMOs
• Genetic modification involves
– mutation, insertion, or deletion of genes.

• Inserted genes usually come from a different species in a form of horizontal gene-transfer.
• In nature this can occur when exogenous DNA penetrates the cell membrane for any reason. This can be
accomplished artificially by: attaching the genes to a virus.

• physically inserting the extra DNA into the nucleus of the intended host with a very small
syringe.

• using electroporation (that is, introducing DNA from one organism into the cell of another
by use of an electric pulse).

• firing small particles from a gene gun.

• Other methods exploit natural forms of gene transfer, such as the ability of Agrobacterium
to transfer genetic material to plants
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•
 Inserting synthetic genes
• Each inserted DNA sequence consists of at least
• a promoter,
• a protein-coding site (the structural gene) and
• a terminator.

• A number of them have been prepared artificially

which include Sugar beet, Chicory, Corn, Cotton, Flax,
Melon, Papaya, Mustard, Potato, Rapeseed, Rice,
Soybean, Squash, Tomato, Barley.
– Others like wheat are under development.
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Alternate Methods of Transforming Plants:
Particle Bombardment

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Agrobacterium-mediated
Transformation

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 Ti Plasmid

1. Large (>200-kb)
2. Conjugative
3. ~10% of plasmid transferred to plant cell
after infection
4. Transferred DNA (called T-DNA) integrates
semi-randomly into nuclear DNA
5. Ti plasmid also encodes:
– proteins involved in mobilizing T-DNA (Vir
genes)

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Ti Plasmid

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Ti Plasmid

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 Only known natural example of DNA
transport between Kingdoms
1. (Virulent) strains
of A. tumefaciens
contain a 200-kb
tumor inducing (Ti)
plasmid

2. Bacteria
transfer a portion T-DNA 
of the plasmid DNA
into the plant host
(T-DNA).
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Agrobacterium
can be used to
transfer DNA
into plants

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VirE2 may get DNA-protein complex across host PM

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Dumas et al., (2001), Proc. Natl. Acad. Sci. USA, 98:485
 Transformation of Arabidopsis plants

Dip floral buds

in 1 ml of
Detergent added to allow Agrobacterium
bacteria to infiltrate the culture for 5 to
floral meristem. 15 min. 22
 Transformation of Arabidopsis plants

700 to 900
seeds per plant.

Germinate on
kanamycin plates
to select
transformants.

10 to 20
transformed
plants per plant.
10 day old seedlings
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 Ti plasmids and the bacterial chromosome act
in concert to transform the plant
1. Agrobacterium tumefaciens chromosomal genes: chvA, chvB,
pscA required for initial binding of the bacterium to the plant cell
and code for polysaccharide on bacterial cell surface.

2. Virulence region (vir) carried on pTi, but not in the transferred

region (T-DNA). Genes code for proteins that prepare the T-
DNA and the bacterium for transfer.

3. T-DNA encodes genes for opine synthesis and for tumor

production.

4. occ (opine catabolism) genes carried on the pTi allow the

bacterium to utilize opines as nutrient. 24
 pTi-based vectors for plant transformation:

1. Shuttle vector is a small E. coli plasmid using for cloning the foreign
gene and transferring to Agrobacterium.

2. Early shuttle vectors integrated into the T-DNA; still produced tumors.

Shuttle plasmid pTi

conjugation
E. coli Agrobacterium
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 Binary vector system
Strategy:
1. Move T-DNA onto a separate, small plasmid.
2. Remove aux and cyt genes.
3. Insert selectable marker (kanamycin resistance) gene in
T-DNA.
4. Vir genes are retained on a separate plasmid.
5. Put foreign gene between T-DNA borders.
6. Co-transform Agrobacterium with both plasmids.
7. Infect plant with the transformed bacteria.

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 Vir (virulent) genes

1. On the Ti plasmid
2. Transfer the T-DNA to plant cell
3. Acetosyringone (AS) (a flavonoid) released by
wounded plant cells activates vir genes.
4. virA,B,C,D,E,F,G (7 complementation
groups, but some have multiple ORFs), span
about 30 kb of Ti plasmid.

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 Vir gene functions (cont.)
• virA - transports AS into bacterium, activates virG post-
translationally (by phosphoryl.)

• virG - promotes transcription of other vir genes

• virD2 - endonuclease/integrase that cuts T- DNA at the

borders but only on one strand; attaches to the 5' end of the
SS

• virE2 - binds SS of T-DNA & can form channels in

artificial membranes

• virE1 - chaperone for virE2

• virD2 & virE2 also have NLSs, gets T-DNA to the nucleus of
plant cell

• virB - operon of 11 proteins, gets T-DNA through 28

Binary vector system

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•Infects at root crown or just below the soil line.

•Can survive independent of plant host in the soil.

•Infects plants through breaks or wounds.

•Common disease of woody shrubs, herbaceous plants,

dicots.

•Galls are spherical wart-like structures similar to tumors

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 The T-DNA is transferred from the
Bacteria into the Nucleus of the Plant
1. Stably integrates (randomly) into the plant
genome.
2. Expression of genes in wild-type T-DNA
results in dramatic physiological changes to
the plant cell.

3.  Synthesis of plant growth hormones

(auxins and cytokinins)  neoplastic growth
(tumor formation)
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Genes required to breakdown opines for use as a nutrient source are
harbored on the Ti plasmid in addition to vir genes essential for the
excision and transport of the T-DNA to the wounded plant cell.

T-DNA 23 kb

bacterial conjugation
tra
pTi
vir genes ~200 kb
for transfer to the plant
opine catabolism

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 Detecting GMOs
• PCR (real time PCR)
– targets the DNA of the plant as well as the DNA of the
promoter which has been used to modify the plant

• ELISA
– depends on assaying the protein which the inserted DNA
sequence is coding for.

• When to use which test

• Nature of the sample
• GMO(s) to be analyzed
• Required test sensitivity
• Whether qualitative or quantitative analysis is required
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• Product’s intended market
 Genetic testing
Broad-spectrum GMO tests:

The same viral and bacterial genetic elements are often incorporated in transgenes to
regulate expression of the trait gene in the plant allowing these DNA sequences to be
targeted and used as broad-spectrum (screening) GMO tests.

These tests are not specific to a particular GM crop or GM event;

each of these broad-spectrum tests can detect many GM crops/events (not all).

If the intent is to determine if GM DNA is “detected” or “not detected” in a sample,

then PCR test(s) targeting one or more broad-spectrum DNA sequences may be
sufficient.

Event-specific and construct-specific GMO tests:

Event-specific and construct-specific PCR assays may be used to identify specific GM

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events.
Additional approaches for molecular marker
analysis / identification
• RFLP
– Restriction fragment length polymorphism
• RAPD
– Random amplified polymorphic DNA
• SSR
– Microsatellite or short sequence repeats
• AFLP
– Amplified fragment length polymorphism
• Mass spectrophotometry
• Electron spray ionization
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 Immunoassays
• RDT
– Advantages: rapid
• ELISA
– Advantages: automated and high throughput

– Overall differences in protein expression levels

may occur in different crop varieties

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 PCR
Can detect all commercialized GMOs
Effective with a broad array of sample types (seed, grain, processed ingredients, finished
products),
Can provide definitive quantification of GMOs since analysis is performed directly at the
DNA level.
Why are polymerases with 3'- 5' exonuclease activity to be avoided in GMO screens
and why is 5' - 3‘ exonuclease activity preferred? Email me the answer

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 Pros and cons of PCR detection
• 1. fore knowledge on inserted DNA
– Else primers can not be designed
• 2. Purpose for detection determines which PCR
technique to use
– Qualitative vs quantitative
– Qualitative / routine screening present or absent
• Genetic control elements such as the cauliflower mosaic virus
35S promoter (P-35S) and the Agrobacterium tumefaciens
nos terminator (nos3') are present in many GMOs currently
on the market (Hemmer, 1997).
– Quantitative how much is present
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 Confirmatory assays
• Gel electrophoresis is the simplest approach to control if the
PCR products have the expected size.

• Southern blot assay, whereby the amplicon is separated by gel

electrophoresis, transferred onto a membrane and hybridised to
a specific DNA probe

• Nested PCR, This strategy reduces substantially the problem of

un-specific amplification, as the probability for the inner pair of
primers of finding complementary sequences within the non-
specific amplification products of the outer pair is extremely low.

• The most reliable way to confirm the authenticity of a PCR

product is its sequencing
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 DNA quality and purity
DNA quality is determined by fragment length and degree of
damage.
This may be caused by exposure to heat, low pH, and/or nucleases
that cause hydrolysis, depurination and/or enzymatic degradation
low quality DNA may contain shorter length fragments, and thus
require primers to target e.g. 100-400 bp
DNA contaminants can include substances that would affect Taq
polymerase
Taq is inhibited by polysaccharides, (EDTA)
ethylenediaminetetraacetic acid phenol, chelex, sodium
dodecylsulfate (SDS), etc
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 Immuno-analysis

• Immunoassays detect proteins that are manufactured in the cell according

to the information coded by the transgenic (GMO) DNA.

• For example, Roundup Ready GM soy has been genetically engineered to

be resistant to the glyphosate herbicide Roundup via insertion of a gene
that codes for a glyphosate tolerant version of a plant enzyme, CP4 epsps.
Once successfully inserted into the soybean genome, the introduced gene
is able to code for the protein enzyme which confers on the plant the
ability to survive treatment with glyphosate.

• Immunological analysis, or immuno-analysis for short, is a GMO test

method that detects proteins. Currently, there are two types of GMO tests
that use this method: the Strip Test and ELISA Method

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 RDT
The assay is rapid, is appropriate for qualitative or semi-quantitative detection of GMO
proteins, and can be performed in the field (at site).
Strip tests are therefore applicable for an initial screen of seed/grain
but not all GMO transgenes code for a protein.
• Therefore, antibody-based tests are not available for all commercialized
GMOs
Absence of both lines indicates that the test is invalid and should be repeated.

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http://www.gmotesting.com/Testing-Options/Immuno-analysis/Strip-Test
 ELISA
The procedure is longer than that of a strip test (hours vs. minutes)

often more sensitive, with a limit of detection in the 0.01 – 1 % range.

has the same requirement for intact protein as the strip test

It is not a suitable GMO

detection method if the
inserted transgene does
not code for protein, or if
proteins in the sample
have been degraded with
heat or chemical
processing.

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http://www.gmotesting.com/Testing-Options/Immuno-analysis/ELISA
Analysis Test Analyte Advantages Disadvantages
Often low sensitivity

operator error can lead to inaccurate test

Rapid results
RDT Protein useful on site
Not appropriate for processed products

Immunologi
c
Not appropriate for processed products
ELIS High sensitivity Must be performed in a laboratory
Protein
A

High sensitivity and

specificity
Capable of detecting all
GMOs
quantitative
Genetic PCR DNA
Must be performed in a laboratory
Effective with broad range
of sample types
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 Advantages and disadvantages
• Although the speed and convenience of antibody-based tests offer substantial utility, a
crucial limitation of these tests is that not all GMO transgenes code for a protein.
Therefore, antibody-based tests are not available for all commercialized GMOs.
Another limitation of strip tests is that the sensitivity is not as great as that of PCR tests.
For example, the limit of detection of strip tests is usually within the 0.1 – 1% range,
whereas the PCR (DNA) test method is more sensitive, with a limit of detection of
0.01%.
•
• Antibody-based tests are also not appropriate for heat or chemically processed
products (e.g., soy protein isolate, lecithin, etc.) in which the protein is denatured
resulting in destruction of the antibody binding site. The protein in processed products
is much more susceptible to degradation than is the DNA; therefore while protein-
based analysis is not appropriate for GMO testing of processed products, DNA-based
PCR analysis may be used for GMO testing of many processed sample types. Finally,
there may be differences in GM protein levels between different commercial GM
cultivars as well as between different parts of the GM plant. All of these factors need to
be taken into consideration when considering antibody-based tests. However, for
seed/grain, in which the protein is intact, GMO strip tests provide an important and
rapid on-site initial screening method. 49
 GMO

• Detection of GMO
– Qualitative screen for presence or absence

• Identification of GMO
– DNA or protein product(s)
– Which particular GMOs are present

• Quantification of GMO
– Exact amount of each GMO present
– Basically for compliance
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 Uses of GMO
• GMOs are used in biological and medical research

• production of pharmaceutical drugs

• experimental medicine (e.g. gene therapy)

• agriculture (e.g. golden rice, resistance to herbicides).

• The term "genetically modified organism" does not always imply, but can
include, targeted insertions of genes from one species into another.
– a gene from a jellyfish, encoding a fluorescent protein called GFP, or green
fluorescent protein, can be physically linked and thus co-expressed with
mammalian genes to identify the location of the protein encoded by the GFP-
tagged gene in the mammalian cell.

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 Detection tools
Validation of all tests pertaining to GMO is imperative and should be specific,
sensitive and have very high detection limits (detect minute amounts)

• Immunological tests

• depends of antigen antibody interaction

• Many of which are conformation depended
• Mainly carried out on raw materials
• Food processing, including heat and acid or alkali
processing can disrupt protein conformation

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 Detection tools continued
• Genetic tests

• Various forms of PCR are used

– Identification
– Detection

• Other emerging tests include

– Mass spec
– DNA chip technology
– Near infrared spectroscopy
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 PCR for GMO identification
• Primer sequence based on specific target transgene sequence

• Some ‘universal’ GMO markers such as

– P-35S Widely used virus resistance genes
– nos3’ (Ti plasmid)

• False positive identification can occur due to P-35S/nos3’ found in

naturally occurring CaMV and A. tumefaciens infections

There are set standard GMO detection methods for each commercially available
GMO.
A GMO register consisting of a database and accompanying monitoring
bioinformatic tools is underway
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e.g. the task force for GM tobacco have set markers
Generic GMO markers

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 Quantification of GMO
• Conventional PCR is not quantitative

• Improved quantitative PCR

– Quantitative competitive PCR Relate Initial template amount
– Real time PCR and final PCR product

– Improved limit of quantification is required

• For 100ng DNA that is 1% GMO, the detection limit corresponds to 0.001%
soyabean, 0.003% maize and 0.02% wheat

– Normalization may be required

• Find amount of GMO relative to non GMO counterpart (reference gene)
• PCR efficiency is essential, co amplification may solve the problem (quantitative
competitive PCR). Assumption that amplification of internal standard will have 56
similar efficiency as target gene
Definitions

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Properties of Taq

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Estimating GMO detection limits

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