Biotechnology:Principlesandprocesses

Whatisbiotechnology?

Biotechnologyreferstothetechnologyusingbiology,whichhas
applicationsinagriculture,foodprocessingindustry,medicine
diagnostics,bioremediation,wastetreatment,andenergyproduction.

TheEuropeanFederationofBiotechnology(EFB)definesbiotechnology
astheintegrationofnaturalscienceandorganisms,cells,parts
thereofandmolecularanaloguesforproductsandservices.

BasisofModernBiotechnology

GeneticengineeringIntroductionofforeigngeneticmaterial
(DNA/RNA)intothehostsgenomeandalteringitsphenotype

AseptictechniquesInvolvesmaintenanceofcontaminationfree
ambienceinchemicalengineeringprocessesformanufactureof
productssuchasantibiotics,vaccines,etc.Thisisdonesoastoenable
thegrowthofonlydesiredmicrobesresponsibleforabioprocess.

GeneticEngineering

Asexualreproductionpreservesthegeneticinformationwhilesexual
reproductionpreservesvariations.

Plantandanimalhybridizationproceduresoftenresultinintroduction
ofundesirablegenesalongwithdesirableones.

Geneticengineeringovercomesthislimitation.

Geneticengineeringincludes:

CreationofrecombinantDNA

Genecloning

Genetransferintohostorganism

TheintroducedpieceofDNAdoesnotreplicateinthehostunlessitis
integratedwiththechromosomeofhost.

Forgettingreplicated,theforeignDNAmustintegrateintothehost
DNAsequencehavingoriginofreplication.Whenthisintegration
occurs,foreignDNAisreplicatedandmanycopiesareformed.This

processiscalledcloning(theprocessofformationofmultipleidentical
copiesofDNA).
ConstructionofaRecombinantDNA

Plasmid(autonomouslyreplicating,circular,extrachromosomalDNA)
isisolated.

PlasmidDNAactsasavectorsinceitisusedtotransferthepieceof
DNAattachedtoittothehost.

PlasmidDNAalsocontainsgenesresponsibleforprovidingantibiotic
resistancetothebacteria.

PlasmidDNAwascutwithaspecificrestrictionenzyme(molecular
scissorsthatcutaDNAatspecificlocations).

TheDNAofinterest(tobeinserted)wasalsocutwiththesame
restrictionenzyme.

TheDNAofinterestishybridisedwiththeplasmidwiththehelpof
DNAligasetoformaRecombinantDNA.

RecombinantDNAisthentransferredtoahostsuchas

E.coli,whereit
replicatesbyusingthehostsreplicatingmachinery.
WhenE.coliisculturedinamediumcontainingantibiotic,onlycells
containingrecombinantDNAwillbeabletosurviveduetoantibiotic
resistancegenesandonewillbeabletoisolatetherecombinants.

RestrictionEnzymesasToolsofRDT

Restrictionenzymesarespecialisedenzymesthatrecogniseandcuta
particularsequenceofDNA.

Nucleasesareoftwotypes:

EndonucleasesCuttheDNAatspecificpositionswithinthe
DNA

ExonucleasesCuttheDNAattheends(Removethe
nucleotidesattheendsoftheDNA)

Everyrestrictionenzymeidentifiesdifferentsequences(Recognition
sequences).Over900restrictionenzymeshavebeenisolated,allof
whichrecognisedifferentsequences.

RecognitionsequencesarepallindromicPallindromesarethe

sequenceofbasepairsthatreadsamebothbackwardsandforwards
(i.e.,same

and

direction).

Example:

Restrictionenzymescutalittleawayfromthecentreofpallindrome
site,butbetweenthesametwobasesontheoppositestrands.

Asaresult,overhangs(calledstickyends)aregeneratedoneach
strand.

Stickyendsformhydrogenbondswiththeircomplementary
counterpartswithhelpofDNAligases.

AlltheseprocessesformthebasisofRDT.

Namingrestrictionenzyme

IstletterGenusoftheorganismfromwhichtheenzymeis
derived

IIndandIIIrdlettersSpeciesoftheorganism

IVthletterNameofthestrain

RomannumberOrderofisolation
E.g.,InEcoRIDerivedfromE.coli,strainR.
ItistheIsttobediscovered.

GelElectrophoresis

Thefragmentsobtainedaftercuttingwithrestrictionenzymesare
separatedbyusinggelelectrophoresis.

Electricfieldisappliedtotheelectrophoresismatrix(commonly
agarosegel)andnegativelychargedDNAfragmentsmovetowardsthe
anode.

Fragmentsseparateaccordingtotheirsizebythesievingpropertiesof
agarosegel.Smallerthefragment,fartheritmoves.

StainingdyessuchasethidiumbromidefollowedbyexposuretoUV

radiationsareusedtovisualisetheDNAfragments.

DNAfragmentsarevisibleasbrightorangecolouredbandsinthe
agarosematrix.

Thesebandsarecutfromtheagarosegelandextractedfromthegel
piece(elution).

DNAfragmentsarepurifiedandthesepurifiedDNAfragmentsareused
inconstructingrecombinantDNAs.

Cloningvectors&hostastoolsofRDT
CloningVectors

Plasmidsandbacteriophagesarecommonlyusedascloningvectors.

Bothofthesehavetheabilitytoreplicatewithinthebacterialcells
independentofthechromosomalDNA.

BacteriophagesHavehighcopynumber(ofgenome)withinthe
bacterialcell

PlasmidsMayhave12copynumberto15100copynumber
percell

IfforeignDNAislinkedtothesevectors,thenitismultipliedtothe
numberequaltothecopynumberofvector.

Featurespresentinthevectoritselfhelpintheeasyisolationof
recombinantsfromthenonrecombinants.

Componentsofaplasmidcloningvector

Originofreplication(ori)

Replicationstartsfromori.AnyfragmentofDNAwhenlinkedto

oricanbemadetoreplicate.

Withthehelpofthis,thegeneticengineermaycontrolcopy
numberoftherecombinantDNA.Torecoverahighnumber,
suitableoriginofreplicationmustbechosen.

Selectablemarker

Thesegeneshelptoselectrecombinantsovernonrecombinants.

AntibioticresistancegenessuchasampR(ampicillinresistant),
tetR(tetracyclineresistant)serveasselectablemarkersusually.

Cloningsites

Thesesitesrefertotherecognitionsitesforrestrictionenzymes
(suchasEcoRI,HindIII,PvuI,BamHI,etc.)

ThesearethesiteswhererestrictionenzymescuttheDNA.

Cloningprocessbecomescompletedwhenmorethanone
recognitionsitesarepresent.

Therefore,ligationiscarriedoutonlyattherestrictionsites
presentontheantibioticresistancegenes.

Howantibioticresistancegeneshelpinselectingrecombinants?

SupposetetRgenehasBamHIrecognitionsite.

WhenBamHIisusedforrestriction,foreignDNAfragmentisinserted
withinthetetRgene.

Hence,tetracyclineresistanceisnotpresentintherecombinants.

Recombinantswillgrowonthemediacontainingampicillin,butwilldie
onmediacontainingtetracycline.

Ontheotherhand,nonrecombinantswillgrowonmediumcontaining
ampicillinaswellasonmediumcontainingtetracycline.

Inthisway,antibioticresistancegenehelpsinselectingtransformants.

Alternateselectablemarker

Otherthanantibioticresistancegenes,alternativemarkerscanbe
used.

Oneofthemisgenecodingfor

galactosidase.

Whenforeigngeneisinsertedwithin

galactosidasegene,the

enzyme

galactosidasegetsinactivated(insertionalinactivation).

Thenthebacteriaaregrownonachromogenicsubstrate.

Nonrecombinantswillproducebluecolouredcolonies.

Recombinantswillproducecolourlesscolonies.

Cloningvectorsforplantsandanimals

Tiplasmid(tumourinducingplasmid)referstotheplasmidof
Agrobacteriumtumefaciens.

A.tumefaciensisaplantpathogen.Itproducestumoursinthe
plantsitinfects.

Tiplasmidcanbemodifiedintoacloningvectorbyremovingthe
genesresponsibleforpathogenicity.

RetrovirusThesearethevirusesthatinfectanimals.Theyproduce
cancersinanimals.

Retrovirusescanbedisarmedtobeusedasacloningvector.

Competenthost

Competenthostreferstothebacterialcellsthathavetheabilityto
takeupthevector(containingRecombinantDNA).

MethodstointroducerecombinantDNAintocompetenthost:

Cellsaretreatedwithdivalentcations(e.g.Ca2+).Then,these
cellsareincubatedwithrecombinantDNAonice,followedby
heatshock(at42),andthenputtingthembackonice.Bythis,
bacteriaareabletotakeuprecombinantDNA.

MicroinjectionRecombinantDNAisdirectlyinjectedintothe
nucleusofanimalcell.

Biolistics(GeneGun)Cellsarebombardedwithhighvelocity
microparticlesofgoldortungsten.

DisarmedvectorasincaseofA.tumefaciensandretrovirus

ProcessesofRDT
IsolationofGeneticMaterial(DNA)

FortheprocessesofRDT,DNAmustbeavailableinitspureform.

Firstofall,cellsaretreatedwithspecificchemicalstobreakopenthe

celltoreleasecellularcomponentssuchasDNA,RNA,proteins,etc.
Thisisdonebyenzymessuchaslysozymes(bacterialcell),cellulase
(plantcell),andchitinase(fungalcell).

ContaminantssuchasRNAandproteinsaredigestedwiththehelpof
ribonucleasesandproteasesrespectively.

AdditionofchilledethanolultimatelyprecipitatesoutthepurifiedDNA,
whichcanbeseenascollectionoffinethreadsinthesuspension.

CuttingofDNAatSpecificLocations

DNAiscutintofragmentswiththehelpofrestrictionenzymes.

Fragmentsgeneratedafterrestrictionareisolatedwiththehelpofgel
electrophoresis.

RecombinantDNAisobtainedbyhybridisinggeneofinterestwith
vector,withthehelpofenzymeDNAligase.

PolymeraseChainReaction(PCR)

RecombinantDNAcanbeamplifiedbyPCR.Severalidenticalcopiesof
itcanbesynthesisedinvitro.

Twosetsofprimers(chemicallysynthesisedoligonucleotidestretches
thatarecomplementarytoaregionofDNA),enzymeDNA
polymerase,anddeoxynucleotidesareadded.

PCRconsistsof3steps:

DenaturationDoublehelicalDNAisdenaturedbyproviding
hightemperature.DNApolymerasedoesnotgetdegradedin
suchhightemperaturessincetheDNApolymeraseusedinthis
reactionisthermostableasitisisolatedfromthermophilic
bacteria,Thermusaquaticus(Taq).

ExtensionReplicationofDNAoccursinvitro.

Thiscycleisrepeatedseveraltimestogenerateupto1billion
identicalcopiesoftheDNA.


InsertionofRecombinantDNAintoCompetentCells

InsertionofrecombinantDNAintohostisdonebyseveralmethods:

Transformationincaseofbacteria

Disarmedvectors,biolistics,andmicroinjectionsincaseofplant
andanimalcells

ThecellsbearingrecombinantDNAareselectedbecausethe
recombinantsexclusivelyhaveselectablemarkerpresentin
them(similartoantibioticresistance).

ObtainingtheForeignGeneProduct

ThisisthestageforwhichtherecombinantDNAwasproduced.

ThecellcontainingrecombinantDNAwillproduceanovelprotein
product(desirableproduct/Recombinantprotein).

Forlargescaleproductionofthedesirableproduct(antibiotics,
vaccines,enzymes),optimumconditionsaretobeprovided.

ContinuouscultureUsedculturemediaisdrainedfromonesideand
freshculturemediaisaddedfromtheotherside.

Cellsarekeptthroughoutintheirlog/exponentialphase.

Largerbiomassisproducedbythismethodleadingtohigher

yield.

BioreactorsLargevesselsinwhichlargevolumes(1001000litres)
ofculturecanbeproduced

Optimalgrowthconditionsformicrobesarepresent
(temperature,pH,substrate,salts,vitamins,etc.).

Abioreactorhasthefollowingcomponentsagitatorsystem,
oxygendeliverysystem,foamcontrolsystem,temperatureand
pHcontrolsystem,samplingports.

DownstreamProcessing

Biosynthesisofmanycompoundssuchasenzymes,alcohols,and
antibioticstakeplacewithinthebioreactor.

Theproductssoobtainedarecrudeandrequireseparation,
purification,andfinishing,whichisdoneunderdownstreamprocessing
(DSP).

DSPmakesacrudebioproductmarketable.

Afterproperseparationandpurification,preservativesareaddedand
thefinishedproductismadetoundergoclinicaltrialsandquality

checksbeforebeingsenttomarket.