Microbiological Methods:

Medium inoculation with

bacterial cells, Growth of
bacterial cells

Instructor: Dr. Shah Nawaz

Why grow organisms in the lab when they are
present in nature?

• Large quantity needed for detailed studies

• To generate strains of interest

• Recombinant protein expression

• Industrial applications
 Culturing Microorganisms
• To grow microorganism of choice in the laboratory
• There are two basic culture techniques used in
microbiology:
1. Liquid culture: bacteria, algae, and some fungi can be reared
in culture tubes (test tubes) in a liquid medium.
Ø Liquid medium is best when you want to rapidly increase
the concentration of the organism or when you want to
grow the cells.
 Culturing Microorganisms
• There are two basic culture techniques used in microbiology:
2. Culture Plates: Liquid medium is solidified using agar
and poured as a thin layer in the bottom of a culture dish
(also sometimes called petri plate)
Ø Culture plates are used when you want to test (1)
antibiotic sensitivity, (2) purified culture (3) isolate
individual colonies from samples.
Colony – macroscopically visible collection of millions of
bacteria originating from a single bacterial cell.
A pure culture is one in which all organisms are descendants of the same organism.
Medium(media, plural): a nutrient blend used to support microbial growth.

• A sterile medium is one which is free of all life forms. It is usually sterilized by heating
it to a temperature at which all contaminating microorganisms are destroyed.

• Colony: is the smallest bacterial unit that can be seen with the naked eye.

• Culture: Is part of specimen grown in cultural media.

• Culture media: is a medium(liquid or solid) that contains nutrients to grow bacteria in

vitro. Because sometimes we cannot identify with microscopical examination directly, and
sometimes we do culture for antibiotics sensitivity testing.

• Prosperities of Media:
•Media has to support the growth of bacteria, should be nutritive (contains the required
amount of nutrients).
•With suitable pH (neutral to slightly alkaline 7.3-7.4).
•Suitable temperature (bacteria grow at 37c).
•Suitable atmosphere.
 Forms Of Culture Media
1) Broth tube: are tubes containing a liquid medium. After
incubation, growth (development of many cells from a few cells)
may be observed as one or a combination of three forms:
Patterns of growth
a) Pellicle: A mass of organisms is floating on top of the broth.
b) Turbidity: The organisms appear as a general cloudiness
throughout the broth.
c) Sediment: A mass of organisms appears as a deposit at the
bottom of the tube.
2) Slant tubes: are tubes containing a nutrient medium plus a
solidifying agent, agar-agar. The medium has been allowed to
solidify at an angle in order to get a flat inoculating surface.
• Slanting the surface of the agar gives the bacteria a greater surface
area on which to grow in a test tube.
2) Agar plates: are sterile petri plates that are aseptically filled with
a melted sterile agar medium and allowed to solidify. Plates are
much less confining than slants and stabs and are commonly used
in the culturing, separating, and counting of microorganisms.
 General protocol for growing bacteria

• Pouring media “plates”

• “Streaking” bacteria on media plates

• “Picking” a “colony” from the plate

• “Inoculating” a liquid culture

Everything needs to be done under sterile conditions!!
Sterile Technique
 Sterile Technique

Ø When culturing bacteria or other microorganisms,

it is important to keep your work area as clean as
possible.
Ø This prevents the introduction of other
microorganisms from the environment into your
culture.
Ø The techniques used to prevent contamination are
referred to as sterile techniques.
 Sterile Technique
1. Start by washing your down your work or
lab benches with a surface disinfectant.
The most commonly used disinfectants for
lab use are:
1. 10% bleach (recommended by the CDC)
2. 85% ethanol
 Sterile Technique

2. Turn off any forced air heating or air

conditioning units that create strong air current in
your work area.
3. A small room or closet that can be closed off is
worth the effort to set-up if you will be doing a
lot of microbial culturing.
4. You can install a UV bulb in a fluorescent light
fixture to surface sterilize your work bench if
you have an enclosed area. Remember to leave
the area when you turn on the UV light source!
 Sterile Technique

5. All glassware should be cleaned and

sterilized before you begin.
6. All pipettes, spatulas, and test tube
(culture) racks should also be sterilized.
7. You can purchase sterile, disposable
culture tubes, petri dishes, and pipettes to
minimize the quantity of glassware that
you have to sterilize.
 Sterile Technique
8. Don’t forget to wash you hands after you finish
cleaning and put on a pair of sterile disposable
gloves before you begin.
9. Once your work area is clean, your hands are
clean, and your glassware is clean and sterile,
don’t contaminate the work area by placing
“dirty items” such as pencils, pens, notes, or
books in the sterile work area.
Media Preparation
 Microbiological Media
• The type of growth medium that you use is a
function of the organisms that you want to
culture. Use a reference book (there are
many) to determine the type of medium that
is best suited for your organism of interest.
• Common media include Luria Broth (LB),
Nutrient Agar, Potato-Dextrose Agar (PDA)
 Luria Broth
Liquid Medium Plate Medium
10 g Tryptone 10 g Tryptone
5 g yeast extract 5 g yeast extract
5 g NaCl 5 g NaCl
Distilled H2O to 1 l volume
Distilled H2O to 1 l volume
2 g Agar
Adjust pH to 7.0
Adjust pH to 7.0
Sterilize for 45 minutes using
Sterilize for 45 minutes using
autoclave or pressure autoclave or pressure
cooker cooker
 How is media made?
• When lab personnel make media they measure out a quantity of dry
powdered nutrient media, add water and check the pH(7).

• They dispense the media into bottles (flask,tube), cap it and autoclave.
The autoclave exposes the media to high temperature (121°C) and
pressure (15 psi) for 20 minutes.

• Once the media is autoclaved it is sterile

• (all microoranism forms killed)
Media Sterilization
 Media Sterilization
There are two reliable methods used to
sterilize microbial culture media:
1. autoclave
2. pressure cooker
When using an autoclave, use the “wet”
setting for sterilizing liquids (flasks, bottles,
culture tubes, etc), and use the “dry” setting
when sterilizing empty containers, stoppers,
etc.
When using a pressure cooker, don’t over fill the
cooker, and remember to weight your containers
so they don’t fall over!
 Plate Pouring Tips
Ø Line empty plates along the edge of the work bench.
Ø Open the petri dish lid at about a 30-45° angle to allow the
hot liquid to cover the bottom of the dish. The thermal
current created by the hot media prevents bacteria and
fungal spores from landing in your clean dish.
 Plate Pouring Tips
Ø As the plates are poured, move the filled plates to
the back of the table until the plates cool and
congeal.
Ø Once the plates have cooled and the media is firm,
store the plates media side-up (bottom) with the
lid securely taped or the plates restacked in the
manufacturer’s plastic sleeve.
Ø To increase the shelf-life of the plates, store in a
cool, dry environment until they are used
(refrigerator).
Inoculating Plates and
Culture Tubes
 Inoculation of Culture Plates and Tubes
ü Use either disposable inoculation loops or a metal loop
that can be heat sterilized to inoculate plates, slants, and
liquid culture tubes.

ü If using a metal loop, be sure to cool the loop by

touching the sterile cooled liquid media or the sterile
culture plate before the placing the loop in your live
culture. Failure to cool the loop will kill your active
microbial cultures!
 Inoculation of Liquid and
Solid (Slant) Culture Tubes
Step 1: Remove the culture tube stopper or
cap with one (do not set it down) and
flame the mouth of the tube to surface
sterilize the mouth. The heated tube
surface will generate a thermal current that
prevents contamination of the culture.
 Inoculation of Liquid and
Solid (Slant) Culture Tubes
Step 2: Without setting any of the culture materials
on the bench, place the sterile inoculation loop
in the culture.
Step 3: Replace cap on the culture tube with the
active microbes and put it in the test tube rack.
Step 4: Without setting the loop down, pick-up a
sterile fresh culture tube with media with one
hand, and remove the cap with the other hand.
 Inoculation of Liquid and
Solid (Slant) Culture Tubes
Step 5: Flame the mouth of the clean culture tube.
Step 6: Place the inoculation loop containing the
microbes in the fresh media and swirl the loop in
the loop in the media to ensure even dispersal in
the media.
Step 7: If using a solid media slant tube, follow
steps 1-5 and then zig-zag the inoculation loop
across the slanted surface of the solid media in
the tube.
 Inoculation of Liquid and
Solid (Slant) Culture Tubes
Step 8: Flame the mouth of the newly inoculated
culture tube and replace the cap.
Step 9: Place the culture tube in test tube rack.
Step 10: Repeat until all of the sterile tubes have
been inoculated. Use a fresh disposable culture
loop for each tube or flame the metal loop after
each tube has been inoculated.
 Inoculation of Liquid and
Solid (Slant) Culture Tubes
Step 11: Incubate the culture at the recommended
temperature (check with your supplier for growth
requirements). If using environmental samples,
incubation at room temperature will avoid the
accidental culture of human pathogens.
Step 12: Dispose of all culture materials in a
biohazard bag and sterilize all old cultures before
pouring out cultures and washing culture tubes.
Disposable culture dishes should be melted in an
autoclave or pressure cooker prior to disposal.
 Inoculating Petri Plates
Step 1:Remove the culture tube stopper or cap with
one (do not set it down) and flame the mouth of
the tube to surface sterilize the mouth. The heated
tube surface will generate a thermal current that
prevents contamination of the culture.
Step 2: Without setting any of the culture materials
on the bench, place the sterile inoculation loop in
the culture.
Step 3: Replace cap on the culture tube with the
active microbes and put it in the test tube rack.
 Inoculating Petri Plates
Step 4: Holding the petri dish lid at an 30-45°
angle, work the inoculating loop from the
outside of the plate toward the center in a
zig-zag pattern that covers approximately
25% of the plate surface (think pie or pizza
slice!).
 Streaking” bacteria on media plates

Streaking loop
Picking a colony and inoculating liquid culture

Put the picked colony

in liquid media to grow
bacteria

Picking a colony
 Inoculating Petri Plates
Step 5: Turn the petri plate 90° to the right, dragging
the inoculation loop through the last section of the
plate, moving from the outside to the inside in a
zig-zag motion.
Step 6: Repeat this process twice more until the
entire plate surface is covered.
NOTE: If you are trying to isolate individual
colonies, each turn of the dish will give you fewer
microbes so that you can distinguish individual
colonies.
 Use of a Plate Counter for
Estimating Microbial Populations
 Serial Dilution of Environmental Samples or
Commercial Cultures
Ø Serial dilution techniques should be used in the estimation
of microbial population sizes.
Ø Serial dilution involves the use of a known amount (in ml
or μl) in a known volume of liquid media.
Ø A one in ten dilution is made in a new liquid culture tube,
and this process is usually repeated several times. The
resulting cultures are dilutions of 1/10, 1/100, 1/1000,
1/10,000, for example, of the original sample.
Ø These cultures are plated on petri plates and incubated at
the recommended temperature.
 Estimating Microbial Population Size
Ø After the inoculated plates are incubated for the
appropriate time period, the number of colonies
per plate are counted.
Ø Population estimates are obtained by multiplying
the dilution factor by the number of colonies per
plate. The resulting number is a rate (function) of
the initial weight or initial volume used from the
environmental sample or culture (per gram soil,
per ml or μl of culture).
 Summary
Different media are used to culture microorganisms,
be certain that you are using the appropriate media for
your organism.
Always use sterile technique to prevent
contamination.
Choose the type of media (liquid or plate) appropriate
for your investigation or application.
Sterile liquid culture tubes and media plates can be
prepared in advance and stored in the refrigerator for
later use (2 weeks for liquid culture tubes, 2 months
for media plates).
 Summary
Liquid culture tubes, solid slant tubes, and
petri plates can be used to culture microbes.
Media and lab materials should be sterilized
prior to use; an autoclave or a pressure cooker
can be used in the sterilization process.
Serial dilution and plate count techniques are
used to estimate microbial populations from
environmental or commercial cultures.
Structure and
function of DNA,
genes and genomes,
cell cycle and cell
division
INSTRUCTOR: DR. SHAH NAWAZ
 Central Dogma
DNA ---------→ RNA---------→Protein.

This unidirectional flow equation represents the Central

Dogma (fundamental law) of molecular biology.

This is the mechanism whereby inherited information is used

to create actual objects, namely enzymes and structural
proteins.

An exception to the central dogma is that certain viruses

(retroviruses) make DNA from RNA using the enzyme
reverse transcriptase.
Gene Expression
Genes are DNA sequences that encode
proteins (the gene product)

Gene expression refers to the process

whereby the information contained in genes
begins to have effects in the cell.

DNA encodes and transmits the genetic

information passed down from parents to
offspring.
 Genetic code
The alphabet of the genetic code contains only
four letters (A,T,G,C).

A number of experiments confirmed that the

genetic code is written in 3-letter words, each of
which codes for particular amino acid.

A nucleic acid word (3 nucleotide letters) is

referred to as a codon.
 Nucleic acids
Principle information molecule in the cell.

All the genetic codes are carried out on the nucleic acids.

Nucleic acid is a linear polymer of nucleotides

Nucleotides
Nucleotides are the unit structure of nucleic acids.
Nucleotides composed of 3 components:
◦ Nitrogenous base (A, C, G, T or U)
◦ Pentose sugar
◦ Phosphate
Nitrogenous bases
There are 2 types:
◦ Purines:
◦ Two ring structure
◦ Adenine (A) and Guanine (G)

• Pyrimidines:
• Single ring structure
• Cytosine (C) and Thymine (T) or Uracil (U).
Nucleotide bases
 Types of Nucleic acids
There are 2 types of nucleic acids:
1. Deoxy-ribonucleic acid (DNA)
◦ Pentose Sugar is deoxyribose (no OH at 2’ position)
◦ Bases are Purines (A, G) and Pyrimidine (C, T).
2. Ribonucleic acid (RNA)
◦ Pentose Sugar is Ribose.
◦ Bases are Purines (A, G) and Pyrimidines (C, U).
 Linear Polymerization of Nucleotides
Nucleic acids are formed
of nucleotide polymers.
Nucleotides polymerize
together by phospho-
diester bonds via
condensation reaction.
The phospho-diester bond
is formed between:
◦ Hydroxyl (OH) group of
the sugar of one
nucleotide.
◦ Phosphate group of other
nucleotide
 Polymerization of Nucleotides
The formed polynucleotide
chain is formed of:
◦ Negative (-ve) charged Sugar-
Phosphate backbone.
◦ Free 5’ phosphate on one end (5’
end)
◦ Free 3’ hydroxyl on other end (3’
end)
◦ Nitrogenous bases are not in the
backbone
◦ Attached to the backbone
◦ Free to pair with nitrogenous bases
of other polynucleotide chain
Polymerization of Nucleotides
Nucleic acids are polymers of nucleotides.
The nucleotides formed of purine or pyrimedine
bases linked to phosphorylated sugars
(nucleotide back bone).
The bases are linked to the pentose sugar to
form Nucleoside.
The nucleotides contain one phosphate group
linked to the 5’ carbon of the nucleoside.
Nucleotide = Nucleoside + Phosphate group
N.B.
•The polymerization of nucleotides to form
nucleic acids occur by condensation reaction
by making phospho-diester bond between 5’
phosphate group of one nucleotide and 3’
hydroxyl group of another nucleotide.

•Polynucleotide chains are always synthesized

in the 5’ to 3’ direction, with a free nucleotide
being added to the 3’ OH group of a growing
chain.
Complementary base
pairing
It is the most important structural feature of
nucleic acids
It connects bases of one polynucleotide
chain (nucleotide polymer) with
complementary bases of other chain
Complementary bases are bonded together
via:
◦ Double hydrogen bond between A and T (DNA),
A and U (RNA) (A═T or A═U)
◦ Triple H-bond between G and C in both DNA or
RNA (G≡C)
Significance of complementary
base pairing
The importance of such complementary base
pairing is that each strand of DNA can act as
template to direct the synthesis of other strand
similar to its complementary one.

Thus nucleic acids are uniquely capable of

directing their own self replication.

The information carried by DNA and RNA

direct the synthesis of specific proteins which
control most cellular activities.
DNA structure
DNA is a double stranded molecule consists of 2
polynucleotide chains running in opposite directions.
Both strands are complementary to each other.
The bases are on the inside of the molecules and the 2
chains are joined together by double H-bond between A
and T and triple H-bond between C and G.
The base pairing is very specific which make the 2
strands complementary to each other.
So each strand contain all the required information for
synthesis (replication) of a new copy to its
complementary.
RNA structure
It is formed of linear polynucleotide
It is generally single stranded
The pentose sugar is Ribose
Uracile (U) replace Thymine (T) in the
pyrimidine bases.

Although RNA is generally single stranded,

intra-molecular H-bond base pairing occur
between complementary bases on the same
molecule (secondary structure)
Types of RNA
Messenger RNA (mRNA):
◦ Carries genetic information copied from DNA in the form of a
series of 3-base code, each of which specifies a particular amino
acid.
Transfer RNA (tRNA):
◦ It is the key that read the code on the mRNA.
◦ Each amino acid has its own tRNA, which binds to it and carries
it to the growing end of a polypeptide chain.
Ribosomal RNA (rRNA):
◦ Associated with a set of proteins to form the ribosomes.
◦ These complex structures, which physically move along the
mRNA molecule, catalyze the assembly of amino acids into
protein chain.
◦ They also bind tRNAs that have the specific amino acids
according to the code.
RNA structure
RNA is a single stranded polynucleotide molecule.

It can take 3 levels of structure;

◦ Primary: sequence of nucleotides
◦ Secondary: hairpin loops (base pairing)
◦ Tertiary: motifs and 3D foldings
RNA structure

Transfer RNA (tRNA) structure

 DNA Part II:

THE CELL CYCLE

“THE LIFE OF A CELL”
AND CELL DIVISION
 Cells & Cell Reproduction

Cells must reproduce else they die. The "life of a cell" is termed the cell cycle.
The cell cycle has distinct phases, which are called G1, S, G2, and M.
Cells that have temporarily or reversibly stopped dividing are said to have
entered a state of quiescence called G0 phase.

72
 The G1 Phase of the Cell Cycle

• During this time organelles

are reproducing, protein
synthesis is occurring for
growth and differentiation.
• Because, transcription is
occurring, the DNA is
uncoiled.
• This phase is the most
variable, ranging from
almost nothing to years.

73
 The G1 Phase of the Cell Cycle

Most cells that differentiate will do

so during this phase. Cells
arrested in G1 may no longer have
the capability of reproducing and
are said to be in G0.
Certain cells in G0, however, when
given some external or internal
cues may revert back to G1 and
enter the cell cycle again.
Nerve and muscle cells are usually
arrested in G0.
 S Phase of the Cell Cycle

The S or synthesis phase is the second

phase of the cell cycle.
•DNA uncoils
•DNA replication occurs
•Additional organelle replication
occurs
•This phase ensures that each
emerging daughter cell will have the
same genetic content as the mother
cell.

75
 G2 Phase of the Cell Cycle
The G2 or Gap 2 phase occupies
the time from the end of S until
the onset of mitosis.
•During this time, the cell
prepares for mitosis by making
and organizing necessary
proteins such as the tubulin
needed to construct
microtubules which used to
make spindle fibers.
•On the average this phase may
take four hours.

76
 M Phase or Mitosis

• During mitosis the nucleus is

replicated and the cytoplasm
divides to produce two
genetically identical daughter
cells.
• Remember that the DNA is
replicated in S prior to mitosis.
• The phases are triggered by the
accumulation of cell signals.
 The Amount of DNA Varies During the Cell Cycle

This graph represents the amount of DNA found in the

cell during the cell cycle. What caused the changes?
What happens at the end of Mitosis?

78
 What Controls the Cell Cycle?

Pay attention to the 3 points in the cycle

that serve as “checkpoints”
79
 Internal Controls of the Cell Cycle

• The control of the cell cycle

is dependent on an
accumulation of “signal
molecules”.
• Quite often these signal
molecules must be
phosphorylated in order to
be functional. This are
simple illustrations.

80
 Cyclins vs. Kinases
Cyclins are a family of proteins that control the progression
of cells through the cell cycle by activating cyclin-dependent
kinase (Cdk) enzymes. Only with the cyclin is the Cdk active.
Cyclins were originally named because their concentration
varies in a cyclical fashion during the cell cycle.

A kinase is a type of enzyme that transfers phosphate groups

from high-energy donor molecules, such as ATP, to specific
substrates, a process referred to as phosphorylation.
.

81
 Cyclins vs. Kinases

• Certain cyclins are made at certain times during the cell cycle, and their
concentration will rise and fall. Cyclins are also destroyed after they are no
longer needed by the cell.

• CDKs are not destroyed as they are only activated or deactivated.

• Which cyclin affects which phase of the cycle? (you don’t have to memorize it
but be able to read it in the graph!)
82
S of the Cell Cycle

83
G2 Phase of the Cell Cycle

84
M Phase or Mitosis
What Controls the Cell Cycle?

86
 Transformation of plants,
transgenic analysis and expression
Instructor: Dr. Shah Nawaz
Developmental Processes 
• Present knowledge of plant hormone and light 
regulation (especially at the molecular level) 
is to a large extent the result of:
 
1) research on Arabidopsis thaliana 

                           and

2) our ability to transform plants using
 the Agrobacterium system.
 Formula of plant
transformation

• Tissue culture + DNA delivery and 
integration = transgenic plants
Methods of delivering DNA into plant cells

•Biological (Indirect methods)

–Agrobacterium–
Other bacteria
–Viruses
•Physical (Direct methods)
–Particle bombardment
–Electroporation 
–Silicon carbide whiskers
–Carbon nanofibers
 History

• The transformation was the first time 
applied by British Bacteriologist 
Frederick Griffith in 1928 in diplococcus 
pneumonia.
• Transformation using electroporation 
was developed in the late 1980s.
• Particle bombardment discovered (gene 
gun) by John Sanford in the 1980s
Particle gun method (Direct
method)
• A biolistic particle or gene gun system 
are designed for the transformation of 
the plant.
• It is a device that injects cells with 
genetic information.
Arabidopsis thaliana
Weed (of no agricultural importance)

Economical reasons to study Arabidopsis:

1) Small size (+/- 30 cm tall at the end of its

life cycle)

2) Short life cycle (+/- 6 weeks from start of

germination to next generation of seeds)

3) Small genome* (complete DNA sequence

is known): 125 million base pairs.

* Combined sequence of all of the chromosomes.
Arabidopsis growth chamber

Up to 1000 individual plants grown to maturity.
 Agrobacterium tumefaciens 
• Plant transformation: inserting a 
piece of foreign DNA into a plant 
chromosome to allow the plant to 
make a foreign protein. 

• Most plant transformation 
technologies use the plant pathogen 
Agrobacterium tumefaciens.
 Crown galls are formed when Agrobacterium
tumefaciens infects wounded plant tissue. 
The wounds often occur around the crown 
(area between stem and root), but can also 
be higher on the stem, like the gall on this 
wallnut tree. The gall tissue grows actively in 
the laboratory. 

Crown galls can be considered the plant 
equivalent of tumors (mammalian 
carcinogenesis).

Fig. 17-5, p. 281

 Genetic engineering by
Agrobacterium tumefaciens

1 Plant tissue is
wounded.

2 Plant secretes acetosyringone, a chemical that

attracts Agrobacterium tumefaciens.
Agrobacterium
3 Bacteria swim to wound and attach to cell walls of
wounded cells.
Ti plasmid
4 Agrobacterium cell injects a specialized piece of
DNA into a plant cell. This DNA fragment is
plant cell
incorporated into a plant chromosome.
nucleus
5 Stimulated by auxin and cytokinin produced by
the enzymes coded in this piece of DNA, the plant
cell repeatedly divides, forming a tumor.

6 The growing tumor serves as a sink for phloem

transport. Nutrients delivered by the phloem are
in part used to make opines, which are secreted.
Bacteria living in the spaces between the plant
cells take up the opines and catabolize them
(break them into components to use for growth).

Fig. 17-7, p. 282

 Transforming a plant cell by 
using Agrobacterium
Gene to be introduced in plant 
cell (for example: a gene that 
encodes the Luciferase protein)
Plant Cell
+ Agrobacterium

Modified 
Nucleus Ti-plasmid
Transformed 
Plant Cell
Agrobacterium
Plant cell makes 
luciferase protein
Example of genetically engineered
plant:Tobacco plant glows in the dark 
because the new gene that was inserted 
(which came from a firefly) produces the 
enzyme luciferase.

By using an appropriate 
cytokinin to auxin ratio (see 
lecture on plant tissue 
culture) we can produce an 
adult plant starting from a 
single cell.

Fig. 17-8, p. 282

 Summary
       
Agrobacterium tumefaciens mediated gene transfer
•Agrobacterium tumefaciens is a natural genetic engineer.
•It is achieved in two ways:
•Co-culture with tissue explants.
•In-plant transformation.
Agrobacterium tumefaciens Characteristics
•Soil-borne, gram – ve, motile, rod-shaped bacteria that are present in the rhizosphere.
•Encodes by a tumor-inducing plasmid that is a large (250kbp) plasmid, which is a vector that transfer T-DNA 
region into the genome of plants host.
•Causes Crown gall disease.
•Have able to transferred bacterial genes into the plant genome.
•Attracted to wound site via chemotaxis in response to chemicals (Sugar and Phenolic molecules: like 
Acetosyringone) released from damaged plant cells.
How to analyze
transgenics?
 Transgenic animals and
cloning, molecular pharming

The last episode!!

Making Transgenic Plants and
Animals
• Why?
1. Study gene function and regulation
2. Generate new organismic tools for other 
fields of research.
3. Cure genetic diseases.
4. Improve agriculture and related raw 
materials. 
5. Generate new systems or sources for 
bioengineered drugs (e.g., use plants 
instead of animals or bacteria).
The organism of choice for mammalian 
genetic engineers.
  - small
  - hardy
  - short life cycle
  - genetics possible
  - many useful strains and tools
The Nobel Prize in Physiology or Medicine, 2007
Mario R. Capecchi, Martin J. Evans and Oliver Smithies
for their discoveries of "principles for introducing specific gene
modifications in mice by the use of embryonic stem cells"

M. Capecchi Sir  M. Evans O. Smithies

Univ. of Utah Cardiff Univ., UK UNC Chapel Hill
The Problem with trying to make 
KOs: Random DNA Integration
• DNA can integrate into the genome by 
homologous (H) or non-homologous 
(N-H) recombination
• Frequency of N-H >> H (by at least 
5000-fold) in mammalian cells
• If you want H integrants, which you 
need for knock-outs, you must have a 
selection scheme for those.
Vector for integrating a transgene by HR 
(i.e., into a specific site) 

tk1 & tk2 -  two copies of a Herpes Simplex Virus thymidine kinase 
gene  (makes cells susceptible to gancyclovir) 
Neo -  neomycin resistance gene
Homologous regions - homologous to the chromosomal target
Transgene - foreign gene
 Example of what happens with N-H recombination 

Transformed cells are neo-resistant, but gancyclovir sensitive.

homol-->
 What happens with HR

If DNA goes in by HR, transformed cells are both neo-resistant and 
gancyclovir-resistant! 

Use double-selection to get only those cells with a homologous 
integration event.
 From Fig. 5.40
To knock-out a 
gene: 1.
KO
1. Insert neo gene 
into the target 
gene.
2. Transform KO  
plasmid into 
embryonic stem
cells.
3. Perform double-
selection to get 
cells with the  KO
homologous  2,3.
integration (neo & 
gangcyclovir 
resistant).
4. Inject cells with 
the knocked-out 
gene into a 
blastocyst.
 How to make a transgenic mouse.

With DNA

(mouse)
Chimeric mouse
(a) If the recipient ES cells are from a brown mouse, and the 
transformed (transgenic) ES cells are injected into a black (female) 
mouse, chimeras are easily identified by their Brown/Black phenotype.

(b) To obtain a completely transgenic KO mouse (where all cells have 
a KO gene), mate the chimera with a black mouse. Some of the 
progeny will be brown (which is dominant), indicating fertilization with a 
germ-line cell (gamete) that ultimately came from a KO-ES cell. Only 
about 50% of the brown progeny mice, however, will have the KO 
allele, because the transgenic ES cell that underwent meiosis to 
produce the germ-line cell was probably heterozygous for the KOed 
gene.

(c) To obtain a homozygous KO mouse (both alleles are KOs), cross 
brown heterozygotes, and ~1/4 of the progeny will be homozygous.
 Not necessarily 3:1

Similar to Fig. 5.41

 Embryonic Stem Cell

Early Fertilization stage Two Cell Stage

Eight Cell Stage Four Cell Stage

 Embryonic Stem Cell

Blastocyst Fetus (Pluripotent)

Adult
 Gene therapy in humans presents 
some formidable problems
• If you could introduce the gene in early 
development (e.g., eggs? or blastocyst) 
might could cure (or partially cure) many  
diseases.
• How to fix them later, as a child, adolescent, 
adult, etc.?
• Transgenic technology + stem cell technology 
= many interesting possibilities
 Pharming
• Pharming, portmanteau of "farming" and 
"pharmaceutical", refers to the use of genetic 
engineering to insert genes that code for useful 
pharmaceuticals into host animals or plants 
that would otherwise not express those genes, 
thus creating a genetically modified organism 
(GMO). Pharming is also known as molecular
farming, molecular pharming or 
biopharming.
• The products of pharming are recombinant 
proteins or their metabolic products. 
Recombinant proteins are most commonly 
produced using bacteria or yeast in a 
bioreactor, but pharming offers the 
advantage to the producer that it does not 
require expensive infrastructure, and cost.
 Pharming as an ethics concern
• In late 2002, just as ProdiGene was ramping up production of 
trypsin for commercial launch it was discovered that volunteer 
plants (left over from the prior harvest) of one of their GM corn 
products were harvested with the conventional soybean crop 
later planted in that field. ProdiGene was fined $250,000 and 
ordered by the USDA to pay over $3 million in cleanup costs. 
This raised a furor and set the pharming field back, dramatically. 
Many companies went bankrupt as companies faced difficulties 
getting permits for field trials and investors fled. In reaction, 
APHIS introduced more strict regulations for pharming field trials 
in the US in 2003. In 2005, Anheuser-Busch threatened to 
boycott rice grown in Missouri because of plans by Ventria 
Bioscience to grow pharm rice in the state. A compromise was 
reached, but Ventria withdrew its permit to plant in Missouri due 
to unrelated circumstances.
The industry has slowly recovered, by focusing on pharming in simple plants 
grown in bioreactors and on growing GM crops in greenhouses. Some 
companies and academic groups have continued with open-field trials of GM 
crops that produce drugs. In 2006 Dow AgroSciences received USDA approval 
to market a vaccine for poultry against Newcastle disease, produced in plant cell 
culture – the first plant-produced vaccine approved in the U.S.
Molecular breeding of plants and
animals, plant tissue culture
Instructor: Dr. Shah nawaz
 What is breeding?
• Modern breeding is an application of 
genetic principles.  
• Crops and animals improvement is a 
cyclic process of identifying new 
variation, crossing, selection, and fixing 
favorable traits.
•   Fundamentally breeding is evolution 
by artificial selection.  
Do we still need to train
students in breeding?
History and development of
breeding
(a journey through time)
 Selected milestones in plant and animal breeding
9000 BC First evidence of plant domestication in the hills above the
Tigris river
1694 Camerarius first to demonstrate sex in (monoecious) plants and suggested
crossing as a method to obtain new plant types
1714 Mather observed natural crossing in maize
1761-1766 Kohlreuter demonstrated that hybrid offspring
received traits from both parents and were intermediate in
most traits, first scientific hybrid in tobacco
1866 Mendel: Experiments in plant hybridization
1900 Mendel’s laws of heredity rediscovered

1944 Avery, MacLeod, McCarty discovered DNA is hereditary

material
1953 Watson, Crick, Wilkins proposed a model for DNA
structure
1970 Borlaug received Nobel Prize for the Green Revolution
Berg, Cohen, and Boyer introduced the recombinant DNA
technology
1994 ‘FlavrSavr’ tomato developed as first GMO
1995 Bt-corn developed
DNA: nucleic acid that 
contains all the 
genetic instructions 
used in the 
development and 
functioning of all 
known living 
organisms

National Human Genome Research 
Institute by Darryl Leja 
 Basic Genetics
• The basic unit of inheritance is the gene, located on 
chromosomes which are made up of strands of 
complex molecules called DNA. 

• In mammals, these chromosomes contain about 60,000 
genes, the entire set of which is called a genome.
• Different forms of the same gene are called alleles. If an 
individual’s two alleles (one from each parent) are the 
same they are homozygous. If different they are 
heterozygous. This genetic makeup is called genotype. 
What can be seen or measured is called phenotype. 
• Progeny are created by the union of gametes (sperm 
from the male and egg from the female). Each gamete 
produced by an individual is unique.  Patterns of 
inheritance depend on:
– The gametes produced by the parents.
– The possible combination of gametes.
– The action of different alleles.
 Types of Inheritance
• Qualitative - One pair or relatively few alleles generally  
involved. Traits have distinct differences in phenotype, 
such as color, horns, etc.  Usually not influenced by the 
environment.
• Quantitative - Many pairs of genes involved, acting 
additively. There is continuous variation in phenotypes. 
Virtually all of the economically important traits result 
from this type of inheritance.  Generally influenced by the 
environment.
Causes of Phenotypic Variation

• Heredity:      
   Animals genetic background for 
phenotype

• Environment:
   Conditions under which the animals are 
born and raised; climate, nutrition, 
disease, general management, etc.  
 Heritability
• Degree of relationship between genotype and 
phenotype. Generally reported as a percentage.  

• Production traits vary in heritability. Most reproductive 
traits (conception rate, etc.) are low (< 20%), 
production traits (weight, etc.) are medium (20% to 
40%), and product traits (lean tenderness, etc.) are 
high (> 40%).  
 • Domestication: The process by which people
try to control the reproductive rates of
animals and plants. Without knowledge on the
transmission of traits from parents to their
offspring.

• Plant Breeding: The application of genetic

Prunus analysis to development of plant lines better
persica
Source: Wikipedia
suited for human purposes.

– Plant Breeding and Selection Methods to meet the

food, feed, fuel, and fiber needs of the world
– Genetic Engineering to increase the effectiveness
and efficiency of plant breeding.
Example: Peach (Prunus persica)
• Originates from China
• Introduced to Persia and the Mediterranean
region along the silk route
• Trade and cultural interaction
  Breeding objectives

• Food (yield and nutritional value), feed,

fibre, pharmaceuticals (plantibodies),
landscape, industrial need (eg. Crops are
being produced in regions to which they are
not native).
• Note: Details among plant species vary
because of origin, mode of
reproduction, ploidy levels, and traits
of greater importance and
adjustments were made to adapt to
specific situations.
Conducting breeding experiments

• Traditional/classical breeding: crossing

two plants (hybridization)
genetically manipulating??
• Variability/ Selection
• Recombinant DNA technology
 Heritability
• Degree of relationship between genotype and 
phenotype. Generally reported as a percentage.  

• Production traits vary in heritability. Most reproductive 
traits (conception rate, etc.) are low (< 20%), 
production traits (weight, etc.) are medium (20% to 
40%), and product traits (lean tenderness, etc.) are 
high (> 40%).  
 SELECTION
• Process that determines which individuals become parents, 
how many offspring they produce, and how long they remain 
in the population.  
• Man practices “artificial” selection.  Nature (the environment) 
practices “natural” selection, which is always present and may 
be the major influence on phenotype in some instances.
• Single trait selection
– Most rapid genetic progress
– May be least adapted to the environment 
• Multiple trait selection
– The more traits the less progress
– Animals raised outside of confinement will need more multiple 
trait selection
 Scientific disciplines and
technologies of plant breeding

• Genetics
• Botany
• Plant physiology
• Agronomy
• Pathology and entomology
• Statistics
• Biochemistry
• Plant tissue culture
 Definition

the culture of plant seeds, organs,

tissues, cells, or protoplasts on
nutrient media under sterile
conditions.
 Basis for Plant Tissue Culture

• Two Hormones Affect Plant Differentiation:

– Auxin: Stimulates Root Development
– Cytokinin: Stimulates Shoot Development
• Generally, the ratio of these two hormones can
determine plant development:
–  Auxin ↓Cytokinin = Root Development
–  Cytokinin ↓Auxin = Shoot Development
– Auxin = Cytokinin = Callus Development
Control of in vitro culture
Cytokinin
Leaf strip

Adventitious
Shoot

Root

Callus Auxin
 Factors Affecting Plant Tissue
Culture
• Growth Media
– Minerals, Growth factors, Carbon source, Hormones
• Environmental Factors
– Light, Temperature, Photoperiod, Sterility, Media
• Explant Source
– Usually, the younger, less differentiated the explant,
the better for tissue culture
• Genetics
– Different species show differences in amenability to
tissue culture
– In many cases, different genotypes within a species
will have variable responses to tissue culture;
response to somatic embryogenesis has been
transferred between melon cultivars through sexual
hybridization
Three Fundamental Abilities of Plants
ü Totipotency
the potential or inherent capacity of a plant cell to
develop into an entire plant if suitably stimulated.
It implies that all the information necessary for
growth and reproduction of the organism is contained
in the cell
ü Dedifferentiation
Capacity of mature cells to return to meristematic
condition and development of a new growing point,
follow by redifferentiation which is the ability to
reorganise into new organ
ü Competency
the endogenous potential of a given cells or tissue to 
develop in a particular way
 Types of In Vitro Culture
ü Culture of intact plants (seed and seedling
culture)
ü Embryo culture (immature embryo culture)
ü Organ culture
1. shoot tip culture
2. root culture
3. leaf culture
4. anther culture
ü Callus culture
ü Cell suspension culture
ü Protoplast culture
 Tissue Culture Applications

ü Micropropagation
ü Germplasm preservation
ü Somaclonal variation
ü dihaploid production
ü Protoplast fusion
ü Secondary metabolites production
ü Genetic engineering
 Genetically Modified Crops

Genetically engineered traits for crop improvement, insect resistance,

herbicide tolerance, abiotic stress tolerance, virus resistance, value added
traits, genetically engineered agricultural crops
 23 years of biotech crops in the world
• Since the first year of commercial planting of biotech crops in 1996, more than 70
countries from all over the world have either planted or imported biotech crops. In
1996, the 6 founder biotech crop countries, the USA, China, Argentina, Canada,
Australia, and Mexico, planted these crops in a total of 1.7 million hectares.
• In 2018, 70 countries adopted biotech crops - 26 countries planted and 44
imported. The total biotech crop area of 191.7 million hectares in 2018 was grown
by 26 countries, 21 developing and 5 industrial countries. Also in 2018, 23 years
after the initial planting, biotech crops increased ~113-fold with an accumulated
global area of 2.5 billion hectares, making biotechnology the fastest adopted crop
technology in the world.
 Genetic Modification vs. Plant
Breeding
• Plant breeding entails successive rounds of
selection of high quality plants with favorable
characteristics.
• Genetic Modification allows individual genes
with desired traits to be moved directly from
one organism into the living DNA of another.
• Genetic engineering advances the science of
specialized crop production by allowing crop
scientists to use genetic traits from species that
are outside of the normal reproductive range of
the plant that is being developed.
What is a Genetically Modified Organism?
• It involves the insertion of DNA from
one organism into another OR
modification of an organism’s DNA
in order to achieve a desired trait.

Suntory "blue" rose

How does this differ from Mendel and his peas?
GM vs Selective Breeding

Selective breeding
• Slow
• Imprecise
• Modification of genes that naturally occur in
the organism

GM
• Very fast
• Precise
• Can introduce genes into an organisms that
would not naturally occur!
Genetic engineering vs agricultural breeding

• Artificial selection has influenced the genetic

makeup of livestock and crops for thousands of
years.
• Proponents of GM crops say GM foods are safe.
• Critics of GM foods say:
– Traditional breeding uses genes from the same
species.
– Selective breeding deals with whole organisms, not
just genes.
– In traditional breeding, genes come together on their
own.
 Agricultural breeding

Traditional breeding changes organisms through

selection, while genetic engineering is more like the
process of mutation.
 GMO in Medicine
• Insulin (e.g., SemBioSys Genetics
Inc- saflower)
• Clotting factors
• Atryn (anticoagulant).
• Banana vaccines
• Cancer fighting eggs
 GMO in Bioremediation

Enviropig i.e., “Frankenswine”

• Poplar trees remove • Able to digest and
groundwater process phosphate
contaminants
 GMO in Pesticides

• Kills caterpillars but

not poisonous to
humans

http://www.nature.com/cr/journal/v12/n2/full/7290120a.html
GMO in Manufacturing

• Produces silk in milk

to make Biosteel
Some genetically modified foods

Bt crops
Some genetically modified foods

Soybean
Roundup ready crops
Abiotic stress tolerence!!

Ice minus strawberries

Some genetically modified foods

Golden rice
Some genetically modified foods

Starlink corn
Some genetically modified foods

AquAdvantage salmon
 Common GM Foods

Products
• Corn
• Canola
• Potatoes
• Tomatoes
• Squash
• Soybeans
• Flax
• Cottonseed oil
• Sugarbeets
Common GM Foods
Genetically Modified Foods
Experts say 60% to 70% of processed
foods on U.S. grocery shelves have
genetically modified ingredients.

Common GM crops:
• Soybeans
• Corn
• Cotton
Genetically Modified Foods
Cons
• Introducing allergens and toxins to food
• Accidental cross pollination
• Antibiotic resistance
• Creation of "super" weeds and other
environmental risks
Genetically Modified Foods
Pros
• Increased pest and disease resistance
• Grow food in harsh climate
• Increased food supply (more food/acre)
• More nutritional value
• Make drugs

Ring spot virus

GMO Controversy in Hawaii

http://hawaiiseed.org/local-issues/taro/
 GMO Controversy in Hawaii
• Undermines the genetic integrity of taro, sacred to the
Hawaiian people;
• Threatens the taro market and livelihood of taro farmers.
Taro production yields over 6 million pounds annually
valued at $3.3 million.
• Threatens the biodiversity of the taro plant;
• Could cause new, unexpected problems in taro
cultivation;
• Could contaminate traditional varieties of taro and take
away taro farmers’ ability to choose what they grow in
their lo’i; and
• Overlooks the wealth of traditional knowledge about
growing taro that has been passed down through
generations.
http://hawaiiseed.org/local-issues/taro/
Recombinant DNA technology,
Genetic mapping, DNA
fingerprinting and MAS
Instructor: Dr. Shah Nawaz

Go to: https://b.socrative.com/login/student/
Remember: Participation in quiz is
compulsory
Happy #DNADay2021! Today commemorates the successful completion of the
Human Genome Project in 2003 and the discovery of DNA’s double helix in 1953,
and celebrates past and future accomplishments in human genetics research. 25th
April 2021
Amplifying DNA

• Need many copies for various DNA tests from a small initial
sample
• Two techniques
• Polymerase chain reaction (PCR)
• Recombinant DNA technology
 PCR

• Done on molecules
• Based on DNA replication
• Rapidly replicates a selected sequence of DNA in a
test tube
• Used to:
• Establish blood relationships
• Identify remains
• Convict or exonerate suspects
• Look at pathogens
• Identify genes
• Very sensitive but easily contaminated
 Review of DNA Replication

A T A T A T A T A T
C G C G C G C G C G
G C G C C G C G C

A T A T A T A T

T A T Nucleotides A T A T A

Parental Both parental Two identical

molecule strands serve daughter molecules
of DNA as templates of DNA
 Recombinant DNA (cloning)

• Works in cells
• Adds genes from one type of organism to the genome of another
• Requires:
• Restriction enzyme
• Vector
• Donor DNA
• Host bacteria
 Restriction Enzymes

Figure 19.3a
Cloning Vectors

• Carries DNA from the cells of one species into cells of another
• Any piece of DNA into which another can be inserted
Types of Vectors

• Which vector used depends on size of the gene to

be inserted
• Plasmid
• One type of cloning vector
• Small circle of double-stranded DNA
• Occurs naturally in some bacteria and yeasts
 Creating Recombinant DNA Molecules

Figure 19.3a
Figure 19.3b
Recombinant DNA

Figure 19.5
Selecting Recombinant DNA Molecules

• 3 types of cells
• Cells that lack plasmids
• Cells that contain plasmids that do not contain a foreign gene
• Cells that contain plasmids that have the foreign gene –WANT THESE
• Plasmids can contain a gene for an enzyme that catalyzes a
reaction that makes a blue color
• If a foreign gene inserts in the middle of this “blue” gene, the
bacteria containing the plasma will not produce the blue color
• Plasmids can contain a gene for antibiotic resistance
• When the antibiotic is applied to the bacterial cells, only those with
the plasmid will survive
Applications of Recombinant DNA
• Drugs (e.g. insulin)
• Pure, human versions
• e.g. “humulin”
• Transgenic plants
• a.k.a. genetically modified (GM) plants
• Transgenic animals
• A transgenic animal is one that carries a foreign gene that has been
deliberately inserted into its genome. The foreign gene is constructed
using recombinant DNA methodology. In addition to the gene itself,
the DNA usually includes other sequences to enable itto be
incorporated into the DNA of the host and
• to be expressed correctly by the cells of the host.
• Transgenic sheep and goats have been produced that express foreign
proteins in their milk.
• Transgenic chickens are now able to synthesize human proteins in the
"white" of their eggs.
Transgenic Plants
Transgenic Animals
Go to: https://b.socrative.com/login/student/
What is gene mapping?

• Genome mapping is used to assign short DNA sequences (molecular

markers) or specific genes to particular regions of chromosomes and
to determine their relative linear orders and distances. A map is an
essential tool for scientists to navigate across the genome.
• Genome maps can be divided into two groups: genetic maps and
physical maps. Genetic maps are based on recombination
frequencies between genetic markers and genes, and linked
markers/genes form linkage groups showing their relative order. A
physical map of a given chromosome or a genome shows the physical
locations of genes and other DNA sequences of interest, and
distances are typically measured in base pair
• Genetic maps have been used successfully to find the gene responsible for
relatively rare, single-gene inherited disorders such as cystic fibrosis and
Duchenne muscular dystrophy. Genetic maps are also useful in guiding
scientists to the many genes that are believed to play a role in the
development of more common disorders such as asthma, heart disease,
diabetes, cancer, and psychiatric conditions
Define genetic markers?

• A genetic marker is a gene or DNA sequence with a known

location on a chromosome that can be used to identify
individuals or species. It can be described as a variation (which
may arise due to mutation or alteration in the genomic loci) that
can be observed. A genetic marker may be a short DNA
sequence, such as a sequence surrounding a single base-pair
change (single nucleotide polymorphism, SNP), or a long one,
like minisatellites.
What are genetic markers?

• Markers themselves usually consist of DNA that does not contain a gene. But
because markers can help a researcher locate a disease-causing gene, they
are extremely valuable for tracking inheritance of traits through generations of
a family.
• The development of easy-to-use genetic maps, coupled with the HGP's
successful sequencing of the entire human genome, has greatly advanced
genetics research. The improved quality of genetic data has reduced the time
required to identify a gene from a period of years to, in many cases, a matter
of months or even weeks.
 Types of markers
List of Markers Acronym
Restriction Fragment Length
RFLP
Polymorphism
Random Amplified Polymorphic DNA RAPD
Amplified Fragment Length
AFLP
Polymorphism
Variable Number Tandem Repeat VNTR
Oligonucleotide Polymorphism OP
Single Nucleotide Polymorphism SNP
Allele Specific Associated Primers ASAP
Inverse Sequence-tagged Repeats ISTR
Inter-retrotransposon Amplified
IRAP
Polymorphism
 Application of DNA markers
Applications of markers in cereal breeding
1.Assessing variability of genetic differences and characteristics within a species.
2.Identification and fingerprinting of genotypes.
3.Estimating distances between species and offspring.
4.Identifying location of QTL's.
5.Identification of DNA sequence from useful candidate genes
Applications for animals
It has 5 applications in fisheries and aquaculture:
1.Species Identification
2.Genetic variation and population structure study in natural populations
3.Comparison between wild and hatchery populations
4.Assessment of demographic bottleneck in natural population
5.markers assisted breeding
Marker assisted selection or marker aided
selection (MAS) is an indirect selection process
where a trait of interest is selected based on
a marker (morphological, biochemical or DNA/RNA
variation) linked to a trait of interest (e.g.
productivity, disease resistance, abiotic stress
tolerance, and quality), rather than on the trait itself.
This process has been extensively researched and
proposed for plant and animal breeding