3 biotechnology basics nelson amugune

8/10/2019 3 Biotechnology Basics Nelson Amugune

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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

3 biotechnology basics nelson amugune

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