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BIOTECHNOLOGY BASICS
Introduction to GMO Biosafety Risk Assessment Course19 -23 OCTOBER, KABANYOLO, UGANDA
Compiled by
Nelson O. Amugune
University of Nairobi, School of Biological Sciences.Email: [email protected] or [email protected]
.
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What is Biotechnology?
Traditional biotechnology
Brewing
oo ermen a on Conventional vaccine production etc
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Modern biotechnology
Cell and tissue culture techniques Plant tissue culture
Animal cell culture
biotechnology Recombinant DNA technology
Monoclonal antibodies Use of diagnostic tools to detect
proteins.
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Scientific disciplines-Molecular Biotechnology
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Why useWhy usebiotechnology?biotechnology?
Increase yield for traditional crops
Improve nutritional and post-harvestqualities of crops
Adapting crops to more stressful
environments Domesticating new crops
Converting existing crops to plantfactories that produce chemicals forindustry
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Application of Biotechnology
Health care
Agriculture, forestry and aquaculture
Industry
Environmental protection Biotechnology for remediation purposes
Alternatives for chemical pesticides
Biotechnology and biodiversity
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PLANT BIOTECHNOLOGY
Requires a good understanding and application ofRequires a good understanding and application of::
Principles of plant breedingPrinciples of plant breeding
GeneticsGenetics
o ecu ar o ogy ano ecu ar o ogy an GenomicsGenomics
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Plant biotechnology
Collaboration from of:Collaboration from of:
Plant breedersPlant breeders
Plant pathologistsPlant pathologists
ys o og s sys o og s s EntomologistsEntomologists
Molecular biologists andMolecular biologists and
GeneticistsGeneticists
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Plant biotechnologyPlant biotechnology
In vitro propagation or tissue cultureIn vitro propagation or tissue culture
DiseaseDisease--free plantsfree plants
Molecular markersMolecular markers
Improved selection in plant breedingImproved selection in plant breeding
Recombinant DNA, to produceRecombinant DNA, to producetransgenic plantstransgenic plants
Use tissue culture techniques
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Tissue cultureTissue culture
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Molecular biology
Enables gene isolation and cloning
Also gene structure
Gene sequence
Development of genetic markers
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Structure of DNAStructure of DNA
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DNA structure (contd)DNA structure (contd)
Linear polymer of 4Linear polymer of 4nucleotides (ACGT)nucleotides (ACGT)
Confi ured as a double helixConfi ured as a double helix
AntiAnti--parallel strands (5parallel strands (5 3)3) Occurs in nucleus tightlyOccurs in nucleus tightly
packed with proteinspacked with proteins
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DNA replicationDNA replication
DNA makes DNADNA makes DNA
By polymerase enzymeBy polymerase enzyme In nucleusIn nucleus
In 5In 5 3 direction onl3 direction onl
Requires beginning (=primer)Requires beginning (=primer)
Involves numerous otherInvolves numerous otherfactorsfactors
e.g DNA ligasee.g DNA ligase
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RNARNA
Single stranded linear polymerSingle stranded linear polymerof 4 nucleotides (ACGU)of 4 nucleotides (ACGU)
Various intermolecularVarious intermolecular
structures possiblestructures possible Different forms with differentDifferent forms with different
functions eg mRNA, rRNA,functions eg mRNA, rRNA,tRNAtRNA
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RNA typesRNA types
mRNA ( messenger RNA)mRNA ( messenger RNA)
Gives proteinGives protein rRNA: ribosomal RNArRNA: ribosomal RNA
Participates in assembly ofParticipates in assembly ofribosomes making proteinribosomes making protein
tRNA: trans er RNAtRNA: trans er RNA Participates in making proteinParticipates in making protein Sn/scRNA: small nuclear/smallSn/scRNA: small nuclear/small
cytoplasmic RNAcytoplasmic RNA Presumed regulatory functionsPresumed regulatory functions
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RNA synthesis:RNA synthesis:
By transcription: DNA makes RNA,By transcription: DNA makes RNA,
Synthesis of RNA by (RNA)Synthesis of RNA by (RNA)polymerasepolymerase
In nucleusIn nucleus
nn -- rec on on yrec on on y
On DNA templateOn DNA template
Requires start and stop signals inRequires start and stop signals intemplatetemplate
Involves numerous other factorsInvolves numerous other factorsand proteinsand proteins
E.g. transcription factorsE.g. transcription factors
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THE STRUCTURE OF A GENE
Eukaryotic genes contain
Introns- segments of DNA that aretranscribed but whose product are
remove rom e ranscr p ur ngmRNA processing
Exons- segments of DNA present in
the mature mRNA and whose productare translated in the cytoplasm
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The structure of a gene (cont'd)
Structural genes - have signals in
front and back i.e. promoters andterminators respectively
romo ers oca e on e s eof DNA. Tells the RNA polymerasewhere to bind and start transcription.
Terminators- On the 3 side. Markthe end of transcription of a structuralgene
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The structure of a gene (contd)
Operons- A genetic unit of transcription
consisting of several structural genes thatare 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 conservedcis-element.
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Gene Structure (cont'd)
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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 andchoose your event(s)
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Expression Vectors
Used to deliver the transgene (gene
of interest) to target tissue/cells
Plasmids and bacterialphages mainly
use Contain selectable markers and
reporter genes.
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Selectable markers
A selectable marker allows preferentialgrowth of transformed cells Mainly antibiotic and/or herbicide
.
The coding sequence usually fused topromoters e.g. nopaline synthase (NOS)and cauliflower mosaic virus (CaMV) 35sregulating sequences
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Antibiotic markers
Neomycin phosphotranferase
type II (NPT II) (kanamycin R)
Hygromycin (HGR)
Ampicillin Streptomycin
Tetracycline
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Rice transformation vector (CAMBIA)
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Reporter Genes
Allow for monitoring of transformation
E.g.
-glucuronidase (GUS) gene
Lac Z gene Luciferase
Anthocyanin
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PLANT TRANSFORMATION
Plant transformation requires
knowledge of both tissue culture andmolecular biology
isolated and cloned into anappropriate expression vector
Regeneration of transformed plant
cells in vitro must be optimized
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Genetic transformationA) Direct gene transfer
Biolistics or particle gun bombardment
Chemicals e.g. PEG (polyethylene glycol)
Microinjection & Macro injection
B) Indirect gene transfer By use ofAgrobacterium
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Agrobacterium-mediated
transformation
Agrobacterium is a gram negative soil
bacterium which harbours Ti plasmid
D-region
(Vir region)
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Transformation (contd)
Wounded plant cells produce phenolic
compounds (eg acetosyringone)which activate the bacterial genes;
e v r reg on co es orendonucleases that cleave the T-DNAhence enabling transfer.
A b t i di t d
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Agrobacterium-mediated
transformation (contd)
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Tumours on passion fruit (Induced
in vivo by wild typeAgrobacterium)
Stem
tumour
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Biolistics
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Transfer to cells
transfection
Virus
Injection (animals)
Transformation
(plants/fish)
Gene gun (plants/fish)
Transgene
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Comparison of DNA delivery
methods
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Expression Vectors
Used to deliver the transgene (gene
of interest) to target tissue/cells Contain selectable markers and
repor er genes.
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Selectable markers
A selectable marker allows preferentialgrowth of transformed cells
Mainly antibiotic and/or herbicide .
The coding sequence usually fused topromoters e.g. nopaline synthase (NOS)and cauliflower mosaic virus (CaMV) 35sregulating sequences
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Antibiotic markers
Neomycin phosphotranferase
type II (NPT II) (kanamycin R) Hygromycin (HGR)
Ampicillin
Streptomycin
Tetracycline
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Reporter Genes
Allow for monitoring of transformation
E.g.
-glucuronidase (GUS) gene
Lac Z gene Luciferase
Anthocyanin
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Gene Expression
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Central DogmaCentral Dogma
DNA makes RNA & RNA makesDNA makes RNA & RNA makesproteinprotein
DNA to DNA: replicationDNA to DNA: replication
DNA to RNA: transcriptionDNA to RNA: transcription RNA to protein: translationRNA to protein: translation
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DNA transcriptionDNA transcription Start signal: promoterStart signal: promoter
Binds on RNA polymerase andBinds on RNA polymerase andtranscription factorstranscription factors
Determines transcriptionalDetermines transcriptionalregulation; is the RNA made, howregulation; is the RNA made, howmuch? Where is made?much? Where is made?
Stop signal: termination ofStop signal: termination oftranscriptiontranscription
In eukaryotes:In eukaryotes: Transcribed RNA is furtherTranscribed RNA is further
modifiedmodified 5 cap, polyA tail5 cap, polyA tail
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Choose and process your gene
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VIRAL INDUCED GENE SILENCING (VIGS)
Virus-induced gene silencing (VIGS) is a gene transcriptsuppression technique for characterizing the function of
plant genes. The approach involves cloning a short sequence of atargeted plant gene into a viral delivery vector.
The vector is used to infect a young plant, and in a few
suppressing virus replication also result in specificdegradation of mRNAs from the endogenous plant gene thatis targeted for silencing.
VIGS is rapid (34 weeks from infection to silencing), doesnot require development of stable transformants, allowscharacterization of phenotypes that might be lethal in stable
lines, and offers the potential to silence either individual ormultiple members of a gene family.
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VIGS (contd)
Apart from VIGS, the most
established technologies used forloss-of-gene function studies in plantsare:
Chemical mutagenesis and The use of transposons or
Agrobacterium T-DNA insertions to
create disruptions in codingsequences.
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THE END
THANK YOU