E. coli plasmids based vectors

E. coli plasmid based vectors and selection strategies for identification of correct recombinant

1. Plasmids
Plasmidsareextrachromosomal,doublestrandedcircularDNAmoleculesthatcarry
anoriginofreplicationandreplicatewithinbacterialcell(originofreplicationisthe
DNAsequenceinagenomeatwhichreplicationisinitiated).
Propertiesofplasmids
a)Integration
Someplasmidsdonotinsertthemselvesintothebacterialchromosome,
theyarecallednon-integrativeplasmids.
Integrativeplasmidsorepisomesareabletoreplicatebyinserting
themselvesintobacterialchromosome.
b)Copynumber
Copynumberisthenumberofmoleculesofanindividualplasmidthat
arenormallyfoundinasinglebacterialcell.Onthebasisofcopy
numberplasmidscanbedividedintotwotypes:
Stringentplasmids:thesehavealowcopynumber,mostlyoneortwo
percell.
Relaxedplasmids:thesearepresentinmultiplecopiesof50ormore
percell.
c)Conjugation
Conjugativeplasmidspromoteconjugationbetweenbacterialcellsthat
canhelptheplasmidtospreadfromonecelltoalltheothercellsina
bacterialculture.(Conjugationandplasmidtransferarecontrolledbya
setoftransferortragenes).
Non-conjugativeplasmidslacktragenesandcannotpromote
conjugation.
d)Compatibility
Differentkindsofplasmidsmaybepresentinasinglecell.For
co-existenceinthesamecell,differentplasmidsmustbecompatible.
Iftwoplasmidsareincompatiblethenoneortheotherwillbelostfrom
thecell.
Typesofplasmidsbasedonmaincharacteristicscoded:
FertilityorFplasmids
ResistanceorRplasmids
ColicinorColplasmids
Degradativeplasmids
Virulenceplasmids
ThebestcharacterizedeukaryoticplasmidthatisfoundinyeastSaccharoyces
cerevisiaeis2µmcircle.

2. CloningVectorsBasedonE.coliPlasmids
A)pBRseries
1)pBR322
p=plasmid,BR=BoliverandRodriguez,thetworesearcherswhodeveloped
pBR322,
322=thenumberdistinguishestheplasmidvectorfromothersdevelopedin
samelaboratory.
Figure:AmapofpBR322
pBR322containsDNAderivedfromthreedifferentnaturallyoccurring
plasmids:
ampRgene–fromplasmidR1,
tetRgene–fromplasmidR6-5
Originofreplication–frompMB1(closelyrelatedtothecolicin-producing
plasmidColE1)
Itisaconjugativeplasmid.
AdvantagesofpBR322:
a)Smallsize:pBR322is4363bp,whichmeansthatthevectoralongwith
recombinantDNAmoleculescanbepurifiedwithease.Evenwith6kbof
additionalDNA,arecombinantpBR322moleculeisstillamanageablesize.
b)Selectablemarkers:itcarriestwosetsofantibioticresistancegenes.Either
ampicillinortetracyclineresistancecanbeusedasaselectablemarkerfor
cellscontainingtheplasmid.
c)Multiplerestrictionsites:
Eachmarkergeneincludesuniquerestrictionsites.

3. InsertionofnewDNAintopBR322thathasbeenrestrictedwithPstI,PvuI,or
ScaIinactivatestheampRgen.
Iinsertionusinganyoneofeightrestrictionendonucleases(notablyBamHI
andHindIII)inactivatestetracyclineresistance.
Thegreatvarietyofrestrictionsitesthatcanbeusedforinsertional
inactivationmeansthatpBR322canbeusedtocloneDNAfragmentswithany
ofseveralkindsofstickyend.
d)Highcopynumber:pBR322hasareasonablyhighcopynumber.Generally
thereareabout15moleculespresentinatransformedE.colicell,butthis
numbercanbeincreased,upto1000–3000,byplasmidamplificationinthe
presenceofaproteinsynthesisinhibitorsuchaschloramphenicol.
2)pBR327
pBR327wasproducedbyremovinga1089bpsegment(frombp1427to2516)
frompBR322.Ithasasizeof3274bp.
ThisdeletionlefttheampRandtetRgenesintact,butchangedthereplicative
andconjugativeabilitiesoftheresultingplasmid.
Asaresult,pBR327differsfrompBR322intwoimportantways:
1)pBR327hasahighercopynumberthanpBR322,about30–45molecules
perE.colicell.ThehighercopynumberofpBR327innormalcellsmakes
thisvectormoresuitableiftheaimoftheexperimentistostudythe
functionoftheclonedgenebecausethemorecopiesthereareofacloned
gene,themorelikelyitisthattheeffectoftheclonedgeneonthehostcell
willbedetectable.
2)ThedeletionalsomakespBR327anon-conjugativeplasmid.Thisis
importantforbiologicalcontainment,preventingtheriskofa
recombinantpBR327moleculeescapingfromthetesttubeandcolonizing
bacteriainthegutofacarelessmolecularbiologist.pBR327is
preferableiftheclonedgeneispotentiallyharmfulshouldanaccident
occur.
Therearemanymorevectorsinthisseries.

4. SelectionstrategyforpBRrecombinants:
EitherampRortetRcanbeusedforselectionofpBRrecombinantsby
insertionalinactivationofeithergene.
BamHIcutswithintheclusterofgenesthatcodeforresistancetotetracycline.
ArecombinantpBR322molecule,thatcarriesDNAinsertintheBamHIsite,
cannolongerprovidetetracyclineresistancetoitshost,asoneofthe
necessarygenesisnowdisruptedbytheinsertedDNA.
CellscontainingthisrecombinantpBR322moleculearestillresistantto
ampicillin,butsensitivetotetracycline(ampRtetS).

5. Figure:SelectionofpBRrecombinants
Aftertransformationthecellsareplatedontoampicillinmediumand
incubateduntilcoloniesappear.Allofthesecoloniesaretransformants
(untransformedcellsareampSandsodonotproducecoloniesontheselective
medium).
Toidentifytherecombinantsthecoloniesarereplicaplatedontotetracycline
containingagarmedium.Afterincubation,someoftheoriginalcolonies
regrow,butothersdonot.Thecoloniesthatdonotgrowontetracyclineagar
arerecombinants(amp
R
tet
S
);oncetheirpositionsareknown,samplesfor
furtherstudycanberecoveredfromtheoriginalampicillinagarplate.

6. B)pUCvectors
p=plasmid,UC=UniversityofCalifornia
pUC8
pUC8isdescendedfrompBR322,itcontainsthereplicationoriginandthe
ampRgeneofpBR322.
TheampRgeneofpUC8doesnotcontaintheuniquerestrictionsites.
AllthecloningsitesareclusteredintoashortsegmentofthelacZ′gene
carriedbypUC8.
Figure:pUCvector
AdvantagesofpUC8
a)Highcopynumber-pUC8hasahighcopynumberof500–700resultingin
highyieldofclonedDNAfromE.colicellstransformedwithrecombinant
pUC8plasmids.
b)Singlesteprecombinantselection-Identificationofrecombinantcellscanbe
achievedbyasinglestepprocess,byplatingontoagarmediumcontaining
ampicillinplusX-gal.AcloningexperimentwithpUC8canthereforebe
carriedoutinhalfthetimeneededwithpBR322orpBR327.
c)DNAwithdifferentstickyendscanbecloned-Therestrictionsitesare
clustered,whichallowsaDNAfragmentwithtwodifferentstickyends(for
exampleEcoRIatoneendandBamHIattheother)tobeclonedwithoutusing
additionalmanipulationssuchaslinkerattachment.
d)SinglestrandedformofGenecanbeobtainedbyM13mpcounterpart
-Therestrictionsiteclustersinthesevectorsarethesameastheclustersinthe
equivalentM13mpseriesofvectors.DNAclonedintoamemberofthepUC

7. seriescanthereforebetransferreddirectlytoitsM13mpcounterpart,enabling
theclonedgenetobeobtainedassingle-strandedDNA.
SelectionforpUCrecombinants(Lacselectionor
α-complementation)
pUC8carriestheampicillinresistancegeneandagenecalledlacZ′,which
codesforpart(α-subunit)oftheenzymeβ-galactosidase.
CloningwithpUC8involvesinsertionalinactivationofthelacZ′gene.
Recombinantsareidentifiedbecauseoftheirinabilitytosynthesize
β-galactosidase.
β-Galactosidaseisinvolvedinthebreakdownoflactosetoglucoseand
galactose.ItisnormallycodedbythegenelacZ,whichresidesontheE.coli
chromosome.
MutanthostE.colihaveamodifiedlacZgene,thatlacksthelacZ′segmentof
β-galactosidase.Thesemutantscansynthesizetheenzymeonlywhenthey
harboraplasmid,suchaspUC8,thatcarriesthemissinglacZ′segmentofthe
gene.
Screeningforβ-galactosidasepresenceinvolvesalactoseanalogcalledX-gal
(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside)whichisbrokendown
byβ-galactosidasetoaproductthatiscoloreddeepblue.

8. Figure:LacSelectionofpUCrecombinants
Figure:LacSelection
AftertransformationthecellsareplatedontomediacontainingX-gal,IPTG
(isopropylthiogalactoside,aninduceroftheenzyme)andampicillin.

9. Thenon-recombinantcolonies(thecellsofwhichsynthesizeβ-galactosidase),
willbecoloredblue,whereasrecombinants(withadisruptedlacZ′geneand
unabletomakeβ-galactosidase),willbewhite.
ThissystemiscalledLacselection.Bothampicillinresistanceandthe
presence/absenceofβ-galactosidasearetestedforonasingleagarplate.
C)pGEM3Zvectors
pGEM3ZisverysimilartoapUCvector.ItcarriestheampRandlacZ′genes.
Itis2750bpinsize.lacZ′regioncontainsaclusterofrestrictionsites.
Figure:amapofpGEM3Zvector(R=clusterofrestrictionsitesforEcoRI,SacI,KpnI,AvaI,
SmaI,BamHI,XbaI,SalI,AccI,HincII,PstI,SphI,andHindIII).
pGEM3Zhastwopromotersequencesthatlieoneithersideoftheclusterof
restrictionsites.Eachpromoteractsastherecognitionsiteforattachmentof
anRNApolymeraseenzyme.
Fig:invitroRNAsynthesis
pGEM3Zandothervectorsofthistypecarryonepromotersthatisspecificfor
theRNApolymerasecodedbyT7bacteriophageandtheotherfortheRNA
polymeraseofSP6phage.TheseRNApolymerasesaresynthesizedduring
infectionofE.coliwithoneorotherofthephages.

10. Theyarechosenforuseininvitrotranscriptionastheyareveryactive
enzymes.TheseRNApolymerasesareabletosynthesize1–2mgofRNAper
minute,substantiallymorethancanbeproducedbythestandardE.coli
enzyme.
AdvantagesofpGEM3Z
ThesevectorscanbeusedforproductionofRNAtranscriptsofdesiredgene.
IfarecombinantpGEM3ZmoleculeismixedwithpurifiedRNApolymerase
inthetesttube,transcriptionoccursandRNAcopiesoftheclonedfragment
aresynthesized.
TheRNAthatisproducedcouldbeusedasahybridizationprobe,ormightbe
requiredforexperimentsaimedatstudyingRNAprocessing(e.g.,theremoval
ofintrons)orproteinsynthesis.
SelectionstrategyforpGEM3Z:sameasforpUCvectors(Lacselection).
REFERENCE:
GeneCloningAndDNAAnalysisAnIntroduction,T.A.Brown,6
th
edition