MichaelFurlong,KristaGladney,MandyPeach PartI:Introduction,ConjugationandTransformation

Introduction:

Bacteria Oneoflargestbiomasses ContributetotherecyclingofN,SandCinecosystems Interactwithhumansex:inmouthsandintestines Implicationsinindustryex:*Lactococcuslactis,cheeseanddairyproducts *Lactococcuslactisisresistanttostreptomycin,tetracyclineandchloramphenicolduetogenesonaplasmid:

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Resistanceisaresultofatwo-stepprocess: 1. Spontaneousmutations 2. Environment SowhatisthesignificanceofthefindingthatresistancegenesinLactococcuslactisarecarriedonaplasmid? Detectingrecombination 1. Workingwithmicroorganism

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2. Phenotypes 3. SelectiveSystems Mechanisms 1. Conjugation 2. Transformation 3. Transduction(Thursday)

DetectingRecombinationInBacteria
Bacteria=Sourceofkeyinsights Whyusebacteria? Smallandsimplegenome Don'tundergomeiosisbutDOrecombineDNA Inbacteria: Genetransferispartialandunidirectional ThereisaDonorandaRecipient Donorfragment=exogenote Recipientgenome=endogenote Cellwithendo+exo=merozygote(partialdiploid)

Specialtechniquesarerequiredforworkingwithmicroorganisms
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Phenotype
Biochemical(ex:wild-typeprototrophic"self-feeding"vsmutantauxotrophic"outside-feeding") Nutritional Resistance Allareanalyzedbygrowthornogrowthunderparticularconditions SelectiveSystems:

Figure3

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BacterialConjugation
RecombinationbythetransferofDNAthroughabridgebetweentwoadjacentcells Dependentonthetransferofthefertilityfactor(F) F+=cellcontainingtheFplasmid F-=cellwithouttheFplasmid ConjugationispossibleduetothemanyimportantpropertiesoftheFplasmid

RecombinationbetweendonorandrecipientDNA
MostofthetimechromosomaltransferisincompleteandstopsbeforeFenters TherecipientwillremainF-butwillacquiresomenewDNAandisnowknownasanexconjugant,containing: 1. thelinearexogenote(fromtheHfrcell) 2. thecircularendogenote(hostgenome) Remember:bacteriacanonlyreplicatecircularDNAlinearDNAdoesnotreplicate!

TheexogenotecanincorporateintotheDNAonlyifadoublecrossoveroccurs

Low-resolutionmappingbyinterruptedconjugation High-resolutionmappingbyrecombinantfrequency

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FFactorsCarryingBacterialGenes

Cleanexcision UseofISsequence Geneincorporation

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Sidenote Eukaryotes=sexgenesfrombothparents Usedgeneticmethodtodetectsexinbacteria TwostrainsofE.coli: StrainY10: + + leu + thi thr StrainY24: bio cys + phe + + grownculturecontainingall6supplements:biotin,cysteine,leucine,phenylalanine,thiamine,threonine cellsplatedandrecombinantgenotypesweredetected

Transformation
"thedirectedmodificationofagenomebytheexternalapplicationofDNAfromacellsofdifferentgenotype" Recipientcellsmustbecompetent Example:transformingDNAfromstrrtorecipientgenotypestrs andplateonstreptomycin FrequencyofresistantcoloniesproportionaltoamountoftransformingDNA

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Figure4:Thegenetictransferofstreptomycin-resistance(strr)tostreptomycin-sensitivity(strs )cellsofE.coli.Therecoveryofstrrtransformants amongstrs cellsdependsontheconcentrationofstrrDNA.

Transformationcanalsobeusedtoassessthetightnessoflinkage IsolationofdonorDNA->randombreakage Iftwogenesareclosetogether: p(breakbetweenthem)low,p(cotransformants)high Iftwogenesarefarapart: p(breakbetweenthem)high,p(cotransformants)stillmoderatebut2genesenteronseparateDNAfragments

PartIConclusions:
1. Bacterialsexualprocessrecombinationofallelesbyassortment 2. Somethingpassingthroughmedium,transferringofhereditarymaterial Pilusgate,notpillar!

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