BIOTECHNOLOGY BASICS

Introduction to GMO Biosafety Risk Assessment Course

19 -23 OCTOBER, KABANYOLO, UGANDA

Compiled by
Nelson O. Amugune
University of Nairobi, School of Biological Sciences.

Email: namugune@uonbi.ac.ke or noamugune@yahoo.co.uk

.

What is Biotechnology?

Traditional biotechnology
 Brewing
 Food fermentation
 Conventional vaccine production etc

Modern biotechnology

Cell and tissue culture techniques
 Plant tissue culture
 Animal cell culture

Genetic engineering/ Molecular

biotechnology
 Recombinant DNA technology

Monoclonal antibodies
 Use of diagnostic tools to detect
proteins.

Scientific disciplines-Molecular Biotechnology

Why use
biotechnology?

Increase yield for traditional crops

Improve nutritional and post-harvest
qualities of crops

Adapting crops to more stressful
environments

Domesticating new crops

Converting existing crops to plant
factories that produce chemicals for
industry

Application of Biotechnology

Health care
Agriculture, forestry and aquaculture
Industry
Environmental protection
 Biotechnology for remediation purposes
 Alternatives for chemical pesticides
 Biotechnology and biodiversity

PLANT BIOTECHNOLOGY

Requires a good understanding and application of






Principles of plant breeding

Genetics
Molecular biology and
Genomics

Plant biotechnology

Collaboration from of:







Plant breeders
Plant pathologists
Physiologists
Entomologists
Molecular biologists and
Geneticists

Plant biotechnology

In vitro propagation or tissue culture
 Disease
Disease--free plants

Molecular markers
 Improved selection in plant breeding

Genetic engineering
 Recombinant DNA, to produce
transgenic plants
 Use tissue culture techniques

Tissue culture

Molecular biology

Enables gene isolation and cloning

Also gene structure
Gene sequence
Development of genetic markers

Structure of DNA

DNA structure (contd)

Linear polymer of 4
nucleotides (ACGT)

Configured as a double helix

Anti
Anti--parallel strands (5 3)

Occurs in nucleus tightly
packed with proteins

DNA replication
DNA makes DNA

By polymerase enzyme
In nucleus
In 5 3 direction only
Requires beginning (=primer)
Involves numerous other
factors
 e.g DNA ligase

RNA

Single stranded linear polymer
of 4 nucleotides (ACGU)

Various intermolecular
structures possible

Different forms with different
functions eg mRNA, rRNA,
tRNA

RNA types

mRNA ( messenger RNA)
 Gives protein

rRNA: ribosomal RNA
 Participates in assembly of
ribosomes making protein

tRNA: transfer RNA
 Participates in making protein

Sn/scRNA: small nuclear/small
cytoplasmic RNA
 Presumed regulatory functions

RNA synthesis:

By transcription: DNA makes RNA,

Synthesis of RNA by (RNA)
polymerase

In nucleus

In 55-3 direction only

On DNA template

Requires start and stop signals in
template

Involves numerous other factors
and proteins
 E.g. transcription factors

THE STRUCTURE OF A GENE

Eukaryotic genes contain

Introns- segments of DNA that are
transcribed but whose product are
removed from the transcript during
mRNA processing

Exons- segments of DNA present in
the mature mRNA and whose product
are translated in the cytoplasm

The structure of a gene (cont'd)

Structural genes - have signals in
front and back i.e. promoters and
terminators respectively

Promoters Located on the 5 side
of DNA. Tells the RNA polymerase
where to bind and start transcription.

Terminators- On the 3 side. Mark
the end of transcription of a structural
gene

The structure of a gene (contd)

Operons- A genetic unit of transcription
consisting of several structural genes that
are transcribed together.
 The operon contains at least two control
regions: the promoter and operator

CIS acting elements within genes help

coordinate gene function.

TATA box- is the most highly conserved
cis-element.

Gene Structure (cont'd)

Making a GMO(Transformation)

Choose your gene

Construct an expression vector
Decide on a vector delivery method
Select the transfected cells
Select your event(s)
Grow into mature organisms, test and
choose your event(s)

Expression Vectors

Used to deliver the transgene (gene
of interest) to target tissue/cells

Plasmids and bacterialphages mainly
used

Contain selectable markers and
reporter genes.

Selectable markers

A selectable marker allows preferential
growth of transformed cells

Mainly antibiotic and/or herbicide
resistance genes.

The coding sequence usually fused to
promoters e.g. nopaline synthase (NOS)
and cauliflower mosaic virus (CaMV) 35s
regulating sequences

Antibiotic markers

Neomycin phosphotranferase
type II (NPT II) (kanamycin R)

Hygromycin (HGR)

Ampicillin

Streptomycin

Tetracycline

Rice transformation vector (CAMBIA)

Reporter Genes
Allow for monitoring of transformation
E.g.

-glucuronidase (GUS) gene

Lac Z gene

Luciferase

Anthocyanin

PLANT TRANSFORMATION

Plant transformation requires
knowledge of both tissue culture and
molecular biology

The gene of interest must first be
isolated and cloned into an
appropriate expression vector

Regeneration of transformed plant
cells in vitro must be optimized

Genetic transformation
A) Direct gene transfer

Biolistics or particle gun bombardment

Chemicals e.g. PEG (polyethylene glycol)
Electroporation
Microinjection & Macro injection

B) Indirect gene transfer

By use of Agrobacterium

Comparison of DNA delivery

methods

Agrobacterium-mediated
transformation
Agrobacterium is a gram negative soil
bacterium which harbours Ti plasmid

D-region
(Vir region)

Ti Plasmid of Agrobacterium
(cont'd)

The LB and RB are the recognition signal
for transfer of the T-DNA; into the plant
genome.

The vir D region contains operons
responsible for the transfer and integration
of T-DNA.

Any insertion/transposon/foreign DNA
present in the T-DNA will be co-transferred
into the plant cell; which may result in
transformation

Transformation (contd)

Wounded plant cells produce phenolic
compounds (eg acetosyringone)
which activate the bacterial genes;

The vir D region codes for
endonucleases that cleave the T-DNA
hence enabling transfer.

Agrobacterium-mediated
transformation (contd)

Tumours on passion fruit (Induced

in vivo by wild type Agrobacterium)

Stem
tumour

Biolistics

Gene gun (plants/fish)

Transfer
to cells
Injection (animals)
transfection

Transformation
(plants/fish)

Virus
(animals/plants)

Transgene

Comparison of DNA delivery

methods

Expression Vectors

Used to deliver the transgene (gene
of interest) to target tissue/cells

Contain selectable markers and
reporter genes.

Selectable markers

A selectable marker allows preferential
growth of transformed cells

Mainly antibiotic and/or herbicide
resistance genes.

The coding sequence usually fused to
promoters e.g. nopaline synthase (NOS)
and cauliflower mosaic virus (CaMV) 35s
regulating sequences

Antibiotic markers

Neomycin phosphotranferase
type II (NPT II) (kanamycin R)

Hygromycin (HGR)

Ampicillin

Streptomycin

Tetracycline

Reporter Genes
Allow for monitoring of transformation
E.g.

-glucuronidase (GUS) gene

Lac Z gene

Luciferase

Anthocyanin

Gene Expression

Central Dogma

DNA makes RNA & RNA makes
protein

 DNA to DNA: replication

 DNA to RNA: transcription
 RNA to protein: translation

DNA transcription

Start signal: promoter
 Binds on RNA polymerase and
transcription factors
 Determines transcriptional
regulation; is the RNA made, how
much? Where is made?

Stop signal: termination of

transcription

In eukaryotes:
 Transcribed RNA is further
modified

5 cap, polyA tail

Choose and process your gene

VIRAL INDUCED GENE SILENCING (VIGS)

Virus-induced gene silencing (VIGS) is a gene transcript

suppression technique for characterizing the function of
plant genes.
The approach involves cloning a short sequence of a
targeted plant gene into a viral delivery vector.
The vector is used to infect a young plant, and in a few
weeks natural defense mechanisms of the plant directed at
suppressing virus replication also result in specific
degradation of mRNAs from the endogenous plant gene that
is targeted for silencing.
VIGS is rapid (34 weeks from infection to silencing), does
not require development of stable transformants, allows
characterization of phenotypes that might be lethal in stable
lines, and offers the potential to silence either individual or
multiple members of a gene family.

VIGS (contd)

Apart from VIGS, the most
established technologies used for
loss-of-gene function studies in plants
are:
 Chemical mutagenesis and
 The use of transposons or
Agrobacterium T-DNA insertions to
create disruptions in coding
sequences.

THE END
THANK YOU