gel electrophoresis purdue university 普渡大学的凝胶电泳

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1、TheEffectsofPlasmidonGenotypeandPhenotype(Revised1/31/96)IntroductionPlasmidsaresmallcircularDNAmoleculesthatoftenfoundinbacteriainadditiontothelargecircularDNAmoleculeofthebacterialchromosome.PlasmidDNAsreplicateindependentlyofthebacterialchromosome,an

2、dmanyplasmidscanalsobetransferrednaturallyamongtheirbacterialhosts.Generally,plasmidgenescodeforproteinsthatarenotessentialforthesurvivaloftheirhostinitsnormalenvironment.However,someplasmidDNAsencodegenesthatconferontheirhosttheabilitytoliveincondition

3、sthatwouldordinarilykillthehost.Forexample,plasmidscanencodeproteinsthatcanbreakdownantibioticsthatwouldotherwisepreventthebacterialcellfromgrowing.Onesuchproteinisanenzymecalledbeta-lactamasewhichcanbreakdownpenicillinandcertainmodifiedpenicillinssucha

4、sampicillin.Ifabacterialcellcontainsaplasmidcarryingthisgene,thenthebacteriacangrowinthepresenceofampicillinandaresaidtohavethephenotype"ampicillin-resistant".TheplasmidyouwillbeusinginyourexperimentsisaplasmidconstructedbyscientistsforuseinrecombinantD

5、NAexperiments.Thebeta-lactamasegenewasincludedinthisplasmidinordertobeabletoeasilytestforitspresenceinbacteriabydeterminingwhethertheycangrowonmediacontainingampicillin.Ageneusedthiswayiscommonlyreferredtoasa"selectablemarker".Sincehavingthisgeneallowsb

6、acteriatogrowinthepresenceoftheantibiotic,youcanreadilyappreciatehowthistypeofgenecancauseseriousmedicalproblemswhenitoccursinpathogenicbacteria.Forthisreason,theplasmidssuchaspUC18whichareusedinrecombinantDNAexperimentsweredesignedsothattheycannotbeexc

7、hangedwithotherbacteriaexceptbyspecialtechniquesusedinthelaboratory.Inthislaboratory,youwillbeincorporatingplasmidsintobacteria(E.coli)andthisprocessiscalledtransformation.ItinvolvestreatingthebacteriainsuchawaythattheircellwallbecomespermeabletoDNAfora

8、periodoftime.ThetransformedbacterialcellswillbeculturedonagarbrothcontainingLuriabroth,ampicillin,andachemicalX-gal(5-bromo-4-chloro-3-indolyl-betaD-galactoside).Luriabrothprovidesessentialnutrientsforgrowthandampicillinwillactas