Screening and selection of recombinants

Screening and selection of recombinants- Direct selection and indirect selection of recombinants

1. SCREENINGANDSELECTIONFORRECOMBINANTS
Dr.Manikandan Kathirvel M.Sc., Ph.D., (NET)
Assistant Professor,
Department of Life Sciences,
Kristu Jayanti College (Autonomous),
(Reaccredited with "A" Grade by NAAC)
Affiliated to BengaluruNorthUniversity,
K. Narayanapura, Kothanur(PO)
Bengaluru 560077

2. SCREENINGANDSELECTIONFORRECOMBINANTS
Theartofcloningistofindtheoneparticulartransformedcell/recombinantcellthatcontainsthecloningvectorwiththe
geneofinterest(referredtoasarecombinantcell).
Itisthusmostimportanttobeabletoselectrecombinantcellsfromtransformedcells(containingthevectorwithout
insertDNA).
IDENTIFICATIONOFRECOMBINANTS:
AftertheintroductionofrDNAintosuitablehostcells,itisessentialtoidentifythosecellswhichhavereceivedtherDNA
molecules.Thisprocessiscalledscreeningorselection.
Generally,theselectionmethodsarebasedontheexpressionornon-expressionofcertaintraitssuchasantibiotic
resistance,expressionofanenzymesuchasβ-galactosidaseorproteinsuchasGFP(GreenFluorescentProtein)and
dependenceorindependenceofanutritionalrequirementsuchastheaminoacidleucine.
Directselection,whichmeansthatthecloningexperimentisdesignedinsuchawaythattheclonesobtainedarethe
clonescontainingtherequiredgene.Almostinvariably,selectionoccursattheplating-outstage.Directselectionisthe
methodofchoice,asitisquickandusuallyunambiguous.However,itisnotapplicabletoallgenes.
Identificationoftheclonefromagenelibrary,whichentailsaninitial“shotgun”cloningexperiment,toproduceaclone
libraryrepresentingallormostofthegenespresentinthecell,followedbyanalysisoftheindividualclonestoidentify
thecorrectone.

3. I)DIRECT SELECTION OF RECOMBINANTS :
Two examples will be discussed:
(i) direct antibiotic resistance screening, and
(ii) blue-white color screening.
II) INDIRECT SELECTION OF RECOMBINANTS :
(i) Screening by Nucleic acid hybridization
(ii) Screening by Colony hybridization
(iii) Screening by Immunological assay
(iv) Screening by Protein/Enzyme activity

4. 1.)Directantibioticresistancescreening
Mostcloningvectorsaredesignedsothattheinsertion
ofaDNAfragmentintothevectordestroystheintegrity
ofoneofthegenespresentonthemolecule(usuallyan
antibioticresistancegene).
INSERTIONALSELECTIONINACTIVATIONMETHOD:
Inthisapproach,oneofthegenetictraitisdisruptedby
insertingforeignDNA.Antibioticresistancegenesactas
agoodinsertioninactivationsystem.
PlasmidpBR322containstwoantibioticresistance
genes,oneforampicillin(amp
r
gene),andtheotherfor
tetracycline(tet
r
gene).
IfthetargetDNAisinsertedintotet
r
geneusingBamHI,
thepropertyofresistancetotetracyclinewillbelost.
Suchrecombinantswouldbetet
s
sensitive.
Whensuchrecombinants(containingtargetDNAintet
r
gene)aregrownintomediumcontainingtetracycline,
theywillnotgrowbecausetheirtet
r
genehasbeen
inactivated.Buttheyareresistanttoampicillinbecause
amp
r
geneisfunctional.
Ontheotherhand,theself-ligatedrecombinantswillshow
resistancetoampicillinandtetracycline.Therefore,theywill
growonmediumcontainingboththeantibiotics.

5. Master plate

6. Bluewhiteselection-
2.VisualScreening:Blue-whitecolourscreening
Amoresophisticatedprocedureforscreeningforthepresenceofrecombinantplasmids,
whichcanbecarriedoutonasingletransformationplate,iscalledblue-whitescreening.
Thismethodalsoinvolvestheinsertionalinactivationofageneandusestheproductionofa
bluecompoundasanindicator.ThegeneislacZ,whichencodestheenzymeß-galactosidase,
andisunderthecontrolofthelacpromoter.
IfthehostE.colistrainisexpressingthelacrepressor,expressionofalacZgeneonthe
vectormaybeinducedusingIPTG(isopropyl-ß-D-thiogalactopyranoside),andtheexpressed
enzymecanutilizethesyntheticsubstrateX-gal(5-bromo-4-chloro-3-indolyl-ß-D-
galactopyranoside)toyieldablueproduct.

7. pUCplasmids:
•pUCplasmidsaresmall,highcopynumberplasmidsofsize2686bp.
•ThisseriesofcloningvectorsweredevelopedbyMessingandco-workersintheUniversityofCalifornia.Thepin
itsnamestandsforplasmidandUCrepresentstheUniversityofCalifornia.
•pUC18andpUC19vectorsareidenticalapartfromthefactthattheMCSisarrangedinoppositeorientation.
•pUCvectorsconsistsoffollowingelements:
►pMB1“rep”repliconregionderivedfromplasmidpBR322withsinglepointmutation(toincreasecopy
number).
►“bla”geneencodingβlactamasewhichprovideampicillinresistancewhichisderivedfrompBR322.Thissiteis
differentfrompBR322bytwopointmutations.
►LacZgenefromE.colilacoperonsystem,whichcontainsMCSrestrictionsites.
•pUCvectorscontainalacZsequenceandmultiplecloningsite(MCS)withinlacZ.Thishelpsinuseofbroad
spectrumofrestrictionendonucleasesandpermitsrapidvisualdetectionofaninsert.
•“rop”geneisremovedfromthisvectorwhichleadstoanincreaseincopynumber.
Copynumber:
500-600copiespercell

8. Alphacomplementation:
Thelacoperonconsistsofstructuralandregulatorygenes.ThelacZgeneisastructuralgeneandisrequired
forgalactosidemetabolism.Thelac-Zgeneproduct(β-galactosidase)isatetramer,andeachmonomerismade
oftwoparts-lacZ-alpha,andlacZomega.
1.ThepBSKS
+
/pUC18plasmidcarriesthealphafragmentofthelacZgene.Thealphafragmentistheamino-
terminusoftheproteinandisnotfunctionalbyitself.ThealphafragmentisdenotedbylacZ'.
2.Typically,theE.colihoststrainusedfortransformationisamutantstrainthathasadeletionofthealpha
fragmentoflacZ.TheE.colichromosomecarriesonlytheomegafragment,whichisthecarboxyl-terminus
oftheprotein.Theomegafragmentaloneisalsonon-functional.
Whenboththealphaandomegafragmentsinteracts,afunctionallyintact-galactosidaseproteinareproduced.
Thisinteractioniscalledalphacomplementation.
Itisdeterminedthatifthealphafragmentwasdeleted,theomegafragmentisnon-functional;however,alpha
fragmentfunctionalitycanberestoredin-transviaplasmid.
Thesubstratesforthebeta-galactosidaseenzymearegalactosides,suchaslactose.Lactoseishydrolyzedby
thisenzymeintogalactoseandglucose.Artificialgalactosides,suchas5-Bromo-4-Chloro-3-Indolyl-beta-D-
galactoside(X-Gal),arealsosubstratesfor-galactosidase.Isopropylthiogalactoside(IPTG)actsasaninducer
oflacZgeneexpression.

9. Cloning in pUC18/19:
Ω-fragment
Vector DNA
GOI
GOI-GENEOF INTEREST
β-galactosidaseenzyme +
chromogenic substrate (X-gal)
Blue color colonies
MCS

10. 1.MultiplecloningsiteisinsertedintothegenelacZ’codingforthealphapeptideofenzymeβ-galactosidase.
(AnMCSisashortDNAsequenceconsistingofrestrictionsitesformanydifferentrestrictionendonucleases).
2.InsertionoftheMCSintothelacZ’fragmentdoesnotaffecttheabilityoftheα-peptide,whilecloningDNA
fragmentsintotheMCSdoes.
3.CloneswithforeignDNAintheMCSdisrupttheabilityofthecellstomakeβ-galactosidase.
4.PlateonmediawithaSubstrateX-gal(β-galactosidaseindicator)andcloneswithintactβ-galactosidase
enzymeindicatethatcellscontainingemptyvectorwithoutinsert,willproducebluecolonies.Colorless
(desirable)coloniesindicatesthatcellscontaintheplasmidwithforeignDNA
5.Therefore,recombinantscanbedetectedbyblue/whitescreeningongrowthmediumcontainingXgalin
presenceofIPTGasaninducer.
Cloning of insert DNA using β -galactosidase: as reporter gene

12. II) Indirect methods:
Identification of the clone from a gene library:
Many different techniques are available for screening a library.
The most important approaches involve
screening by nucleic acid hybridization and
screening by Colony hybridization
screening by functional analysis-Immunological assay
-Protein Assay

13. ScreeningbyNucleicacidhybridization
1.Thefirststepofahybridizationscreeningexperiment
involvesDNAfromtheclonesareisolatedand
separatedontheagarosegelelectrophoresis.The
transferoftheDNAfromthegeltoanylonor
nitrocellulosemembraneiscarriedout.
2.TheDNAisdenaturedwithalkalitoproducesingle
strandsthatareboundedtothemembranebyheat
treatmentorUVirradiation.
3.Themembraneisthenimmersedinasolution
containinganucleicacidprobe(usuallyradioactively
labeled)andincubatedtoallowtheprobetohybridize
toitscomplementarysequence.
4.Afterhybridization,themembraneiswashedto
removeunhybridizedprobe,andregionswherethe
probehashybridizedarevisualizedwith
autoradiography.

14. DNAhybridization.
(1)TheDNAofsamplescontainingtheputativetargetDNAis
denatured,andthesinglestrandsarekeptapart,usually
bybindingthemtoasolidsupport,suchasa
nitrocelluloseornylonmembrane.
(2)(2)Theprobe,whichisoften100to1,000bpinlength,is
labeled,denatured,andmixedwiththedenatured
putativetargetDNAunderhybridizationconditions.
(3)(3)Afterthehybridizationreaction,themembraneis
washedtoremovenonhybridizedprobeDNAand
assayedforthepresenceofanyhybridizedlabeledtag.
(4)Iftheprobedoesnothybridize,nolabelisdetected.
(5)Theasterisksdenotethelabeledtags(signal)ofthe
probeDNA.

15. ScreeningbyColonyhybridization:
ScreeningalibrarywithalabeledDNAprobe(colony
hybridization).
Cellsfromthetransformationreactionareplatedontosolidagar
mediumunderconditionsthatpermittransformed,butnot
nontransformed,cellstogrow.
(1)Fromeachdiscretecolonyformedonthemasterplate,asample
istransferredtoasolidmatrix,suchasanitrocelluloseornylon
membrane.Thepatternofthecoloniesonthemasterplateis
retainedonthematrix.
(2)Thecellsonthematrixarelysed,andthereleasedDNAis
denatured,deproteinized,andirreversiblyboundtothematrix.
(3)AlabeledDNAprobeisaddedtothematrixunderhybridization
conditions.Afterthenonhybridizedprobemoleculesarewashed
away,thematrixisprocessedbyautoradiographytodetermine
whichcellshaveboundlabeledDNA.
(4)Acolonyonthemasterplatethatcorrespondstotheregionof
positiveresponseontheX-rayfilmisidentified.Cellsfromthe
positivecolonyonthemasterplatearesubculturedbecausethey
maycarrythedesiredplasmid-clonedDNAconstruct.

16. Screening by Immunological assay
The desired gene can also be identified by the activities of
the encoded gene product.
1.Thecellsaregrownascoloniesonmasterplateswhich
aretransferredtoasolidmatrix(i.e.,nitrocellulose).
2.Thecoloniesarethensubjectedtolysisandthereleased
proteinsboundtothematrix.Theseproteinsarethen
treatedwithaprimaryantibodywhichspecificallybinds
totheprotein(actsasanantigen),encodedbythetarget
DNA.
3.Afterremovingtheunboundantibodybywashings,a
secondantibodyisaddedwhichspecificallybindstothe
firstantibody.
4.Againtheunboundantibodiesareremovedbywashings.
Thesecondantibodycarriesanenzymelabel(e.g.,horse
reddishperoxidaseoralkalinephosphatase)boundtoit.
Thedetectionprocessissodevisedthatasacolourless
substrateitisacteduponbythisenzyme,acoloured
productisformed.
5.Thecolonieswhichgivepositiveresult(i.e.,coloured
spots)areidentified.Thecellsofaspecificcolonycanbe
sub-culturedfromthemasterplate.

17. ScreeningbyProteinactivity
DNAhybridizationandimmunologicalassaysworkwellfor
manykindsofgenesandgeneproducts.
1.Ifthetargetgeneproducesanenzymethatisnot
normallymadebythehostcell,adirect(insitu)plate
assaycanbedevisedtoidentifymembersofalibrary
thatcarrytheparticulargeneencodingthatenzyme.
2.Thegenesforα-amylase,endoglucanase,β-glucosidase,
andmanyotherenzymesfromvariousorganismshave
beenisolatedinthisway.
3.Thisapproachhasproveneffectiveforisolatinggenes
encodingbiotechnologicallyusefulenzymesfrom
microorganismspresentinenvironmentalsamples.
4.Thistechniquehasenabledtheisolationofmanynovel
proteinswithinterestingpropertieswithouttheneedto
firstculturethenaturalhostmicroorganism.
Screeningagenomiclibraryforenzyme
activity.
1.Cellsofagenomiclibraryareplatedontosolid
mediumcontainingthesubstrateforthe
enzymeofinterest.
2.Ifafunctionalenzymeisproducedbyacolony
thatcarriesaclonedgeneencodingthe
enzyme,thesubstrateisconvertedtoa
coloredproductthatcanbeeasilydetected.
3.Notethatother,noncoloredcoloniesonthe
mediumalsocontainfragmentsofthegenomic
library,buttheydonotcarrythegeneforthe
enzymeofinterest.