• Includinganoriginofreplication(i.e.theE.colioriCregion)intoacircularDNAmoleculeisamechanismtohaveanextrachromosomalelementintheprokaryoticcell.

• Suchanextrachromosomalelementiscalledaplasmid,orvector
• Theplasmidusesthehostcellmachinery(i.e.polymerases,helicases,dNTP"setc.)todirectreplication.

• However,sincetheaddedworkofreplicatingtheextrachromosomalelementisaloadonacell,itwillbeout-competedbyothercellswhichdonotcontaintheplasmid.
  • Sinceinprokaryoticcellsthesegregationofplasmidsisarandomevent,daughtercellscanarisewhichdonotcontaintheplasmidandthesegrowfaster(out-compete)theparentcell.
  • Inotherwords,intheabsenceofotherpressures,afteraperiodoftimethepopulationofcellsinaculturewillbethosewhichhave"lost"theplasmid.

• Inorganismswithmorethanonechromosome(eukaryotes)thereareavarietyofmechanismstoensurethatpropersegregationofchromosomesoccurs,i.e.tomakesurethatdaughtercellscontainequalnumbersofallthechromosomes.

• Onebasicmechanismisthateachchromosomecontainsessentialgenes,andifthesearelost,thecellcannotsurvive.

• Byfarthemostcommonapproachtothemaintenanceofplasmidsisthroughtheincorporationofdrugresistancegenes.
  • Thesearealsoknownasselectablemarkers,i.e.wecanselectfortheirpresencebyincludingantibioticsinthegrowthmedia.

• Ampicillinbindstoandinhibitsanumberofenzymesinthebacterialmembranethatareinvolvedinthesynthesisofthegramnegativecellwall.
  • Therefore,propercellreplicationcannotoccurinthepresenceofampicillin.
• Theampicillinresistancegene(amp^r)codesforanenzyme(b-lactamase)thatissecretedintotheperiplasmicspaceofthebacteriumwhereitcatalyzeshydrolysisoftheb-lactamringoftheampicillin.
  • Thus,thegeneproductoftheamp^rgenedestroystheantibiotic.
• Overtimetheampicillininaculturemediumorpetriplatemaybesubstantiallydestroyedbyb-lactamase.
  • Whenthisoccurs,cellpopulationscanarisewhichhave"lost"theplasmid.

Tetracycline

• Tetracyclinebindstoaproteinofthe30SsubunitoftheribosomeandinhibitsribosomaltranslocationalongthemessengerRNAwhichcodesfortheprotein(i.e.thedruginterfereswithnormaltranslationorproductionofproteins).
  • Thetetracyclineresistancegene(tet^r)encodesa399aminoacidoutermembraneassociatedproteinofgramnegativecellsthatpreventstheantibioticfromenteringthecell.
• Thus,thisdrugresistancegenedoesnotdestroytheantibiotic.Pressurewillbemaintainedthroughoutthecellcultureprocesstokeeptheplasmidcontainingthedrugresistantgene.

• Chloramphenicolbindstotheribosomal50Ssubunitandinhibitsproteinsynthesis.
• Thechloramphenicolresistancegene(Cm^r)codesforaproteinknownatthecatprotein.
  • Thecatproteinisatetramericcytosolicproteinthat,inthepresenceofacetylcoenzymeA,catalyzestheformationofhydroxylacetoxyderivativesofchloramphenicolthatareunabletobindtotheribosome.
  • Likewithampicillin,theCm^rgeneproductdestroystheantibiotic.
  • Additionally,theexpressionofcatproteinisinfluenced(downregulated)bythepresenceofglucoseinthemedia.

• Bindtoribosomalcomponentsandinhibitsproteinsynthesis.
• TheKan^rgenecodesforaproteinwhichissecretedintotheperiplasmicspaceandinterfereswiththetransportoftheseantibioticsintothecell.
  • Liketetracyclineresistance,theKan^rgenedoesnotdestroytheantibiotic.

• Thisisamemberofageneralclassofsubstancesknownasbacteriocins.
• ColicinE1causeslethalmembranechangesinbacteria.
  • Thedrugresistancegene(cea)codesforaproteinthatinterfereswiththeactionofcolicininanunknownmanner.

• InadditiontomakingaplasmidbyusingtheE.coliOriCregion,thereisanaturallyoccuringE.coliplasmidcalledtheColE1plasmid
  • TheColE1originofreplicationisuni-directional(unlikeoriC)
  • ThereplicationfromtheColE1oriregiondoesnotrequiretheassociatedproteins(e.g.dnaAprotein)likeoriC,(butitdoesrequirespecificRNAmolecules).
• DependingontheexactregionoftheColE1originwhichisinsertedintoacirculardnamolecule,theextrachromosomalelementwillbemaintainedwitheithera"low"or"high"copynumber
• TheropgeneneartheColE1originisinvolvedintheregulationofreplication.
  • IftheColE1oriregionincludesthisgenethentheplasmidismaintainedwithanaveragecopynumberof10-30plasmids/cell.Thisisconsideredlowcopynumber.
  • IftheColE1oriregiondoesnothavetheropgene,thentheresultingplasmidismaintainedwithanaveragecopynumberof100-200plasmids/cell.Thisisconsideredahighcopynumbertypeofplasmid.
• Iftheplasmidcontainsagenecodingforaprotein(likedrugresistancegenes)copynumbercaninfluencetheamountofsuchproteininthecell.

• Oneoftheoriginalcloningplasmids.
• Constructedbyligatingtogether:
  • thetetracyclineresistancegenefromplasmidpSC101
  • ColE1andropregionfromtheColE1plasmid
  • theampicillinresistancegenefromthetransposonTn3
• Containsbothampicillinandtetracyclineresistancegenes(markers).
• Containsuniquerestrictionsitesinsideandoutsideofthesemarkers.
• ContainsropregionnearCoE1ori,therefore,ithasalowcopynumber(10-30)
• NumberingbeginsattheuniqueEcoRIrestrictionsite(GAATTC).Thefirst"T"inthissequenceisbasenumber"1".

Theplasmiddiagram:

• Inthecenteristheplasmidname(usuallystartswithalowercase"p")andthesizeinbasepairs
• theinnerringprovidesticksin1Kilobase(Kb)intervalstogiveanideaofthegenerallocationofpartsoftheplasmid
• thearrowsindicategenes,markers,oriorreplication,promoters,polylinkers,transcriptionterminators,andotherimportantorfunctionalitems
• theouterringusuallyindicatesthelocationofunique,orlimitednumber(usually<3),restrictionendonucleasesites.Restrictionenzymeswhichhavemorethanthreesiteswillnotbeindicated.Beawarethatenzymeswhichdonotcutatallwillalsonotbelisted!

• LackstheropgeneneartheColE1oriregion.Thus,thisplasmidtendstoaccumulateinhighcopynumber(100-200).
• Thisvectorcontainsonlytheampicillinresistancemarker.
• Thisvectorcontainsapolylinkerregion
  • AsyntheticDNAsequencewhichcontainsaclusteringofuniquerestrictionenzymerecognitionsites
  • AllowsDNAfragments,generatedbyavarietyofrestrictionendonucleasecleavages,tobeinsertedintotheplasmid
  • pUC18hasthepolylinkerinoneorientation
  • pUC19hasthesamepolylinker,butintheoppositeorientation
  • Thus,fragmentswithuniquerestrictionsitesoneachendcanbeinsertedinaspecificorientation
  • ThePstIsiteintheamp^rgenewasmutatedtoremoveit.TheEcoRIsiteatposition(1)wasmutatedtoremoveit.Thiswasdonetomaketherestrictionsitesinthepolylinkerregionunique
• Thisvectoralsocontainsatranscriptionpromoterregionfromthelacoperon,whichallowsforeigngenestobeinsertedandtranscribed/translated.
  • Thepolylinkerregionisjustdownstream(3")tothelacpromoter
  • Insertedgenescanbetranscribedfromthispromoter