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BIOB12H3 (30)
Lecture

BIOB12H3S-2014-PDF#6.pdf

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Department
Biological Sciences
Course
BIOB12H3
Professor
Aarti Ashok
Semester
Winter

Description
▯ BIOB12H3S:▯Cell▯&▯Molecular▯Biology▯Laboratory▯▯ ▯ Molecular▯biology▯module▯2:▯Gene▯regulation▯(PDF▯#6)▯ ▯ Gene▯regulation▯in▯bacteria:▯the▯lac▯operon▯ Whether▯in▯prokaryotes▯or▯eukaryotes,▯transcription▯involves▯trans▯acting▯factors▯ (e.g.▯RNA▯polymerase)▯moving▯to▯and▯interacting▯with▯specific▯DNA▯sequences▯(cis▯ acting▯sequences)▯to▯influence▯gene▯expression.▯We▯now▯know▯that▯there▯are▯a▯wide▯ variety▯of▯sequence▯specific▯DNA▯binding▯proteins▯that▯coordinate▯transcription▯of▯ specific▯genes▯or▯sets▯of▯genes▯to▯alter▯the▯metabolism▯of▯the▯cell▯or▯organism.▯▯ Typically,▯the▯control▯sequences▯lie▯‘upstream’▯from▯the▯gene▯in▯a▯region▯called▯the▯ promoter.▯As▯has▯been▯discussed▯in▯other▯courses▯you▯have▯taken,▯such▯transcription▯ factors▯often▯act▯in▯combination▯to▯control▯the▯expression▯of▯the▯gene).▯Some▯are▯ negative▯regulators▯(repressors)▯and▯some▯are▯positive▯regulators▯(inducers).▯Often,▯ very▯rapid▯changes▯can▯occur▯in▯response▯to▯changes▯in▯the▯environment▯of▯the▯cell.▯ Responses▯to▯the▯presence▯(or▯absence)▯of▯hormones,▯growth▯factors,▯and▯other▯ metabolites▯as▯well▯as▯changes▯in▯physical▯parameters▯such▯as▯temperature▯can▯quite▯ dramatically▯alter▯signal▯transduction▯to▯condition▯an▯appropriate▯response.▯▯ ▯ Because▯they▯are▯relatively▯simple▯systems,▯prokaryotes▯have▯been▯extensively▯ studied▯by▯molecular▯biologists,▯and▯the▯lactose▯promoter▯is▯arguably▯the▯most▯well▯ characterized▯operon:▯a▯cluster▯of▯structural▯genes▯regulated▯by▯a▯single▯promoter.▯ The▯lac▯operon▯coordinates▯the▯catabolism▯of▯lactose▯into▯the▯simpler▯sugars▯ galactose▯and▯glucose▯(the▯preferred▯carbon▯source).▯▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ β-galactosidase ▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ 1 The▯lac▯operon▯encodes▯3▯genes,▯genetically▯referred▯to▯as▯lac▯Z,▯lac▯Y▯and▯lac▯A.▯▯ ▯ ▯ Lac I promoter Lac promoter ▯ Repressor gene Structural genes ▯ Operator ▯ ▯ P lac I lac P lac O lac Z lac Y lac A ▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯▯▯Lactose▯ ▯ ▯ ▯ β▯galactosidase▯ ▯ Galactose▯+▯Glucose▯ ▯ ▯ Glactoside▯permease▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ Thiogalactoside▯▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ transacetylase▯ ▯ ▯ The▯lac▯I▯gene▯encodes▯the▯lac▯repressor,▯which▯is▯constitutively▯expressed.▯▯In▯the▯ absence▯of▯lactose,▯the▯repressor▯protein▯binds▯to▯the▯operator▯sequence▯and▯blocks▯ the▯transcription▯of▯the▯genes.▯▯Thus▯the▯operon▯is▯off.▯▯When▯lactose▯is▯present,▯it▯ binds▯to▯the▯repressor▯protein▯to▯prevent▯it▯from▯binding▯to▯the▯operator.▯▯Thus,▯there▯ is▯no▯block▯to▯RNA▯polymerase,▯and▯the▯operon▯is▯on.▯Or▯is▯it?▯▯The▯lac▯operon▯is▯also▯ under▯positive▯control▯mediated▯by▯glucose/cAMP.▯▯The▯biochemical▯logic▯is▯this:▯ since▯glucose▯is▯the▯preferred▯carbon▯source,▯why▯go▯to▯the▯energetic▯expense▯to▯ breakdown▯lactose▯into▯glucose▯if▯it▯is▯already▯available?▯Only▯when▯lactose▯is▯present▯ and▯glucose▯concentrations▯are▯low▯does▯it▯make▯sense▯to▯activate▯the▯operon▯to▯ generate▯the▯glucose.▯Thus,▯the▯lac▯operon▯is▯under▯both▯positive▯and▯negative▯ control.▯The▯basic▯facts▯about▯the▯operon▯were▯elucidated▯by▯Jacob▯&▯Monod▯and▯for▯ this▯pioneering▯work▯were▯awarded▯the▯Nobel▯Prize▯in▯1965.▯▯ ▯ In▯this▯lab,▯you▯will▯utilize▯some▯genetic▯tools▯to▯discover▯for▯yourself▯how▯the▯lac▯ operon▯works.▯We▯will▯use▯wildtype▯E.▯coli▯and▯mutants▯in▯the▯lac▯I▯and▯lac▯Z▯genes▯to▯ ask▯questions▯about▯the▯activity▯of▯the▯operon.▯▯How▯does▯one▯know▯when▯the▯operon▯ is▯active?▯One▯way▯is▯to▯measure▯the▯concentration▯or▯activity▯of▯one▯of▯the▯enzymes▯ encoded▯by▯the▯structural▯genes.▯▯Scientists▯have▯been▯quite▯clever▯about▯devising▯ ways▯to▯do▯this,▯not▯to▯investigate▯when▯the▯operon▯is▯active▯but▯to▯enable▯them▯to▯ have▯a▯reporter▯of▯gene▯activity.▯ ▯ β▯galactosidase▯naturally▯recognizes▯its▯normal▯substrate,▯lactose,▯but▯in▯addition,▯it▯ recognizes▯structurally▯related▯molecules.▯Two▯of▯these▯bear▯some▯elaboration.▯Both▯ can▯be▯classified▯as▯chromogenic▯substrates.▯▯That▯is,▯when▯cleaved▯they▯generate▯a▯ product▯that▯is▯colored▯and▯precipitates.▯▯ ▯ 2 5▯bromo▯4▯chloro▯3▯indolyl▯b▯galactopyranoside▯(also▯known▯as▯X▯GAL)▯gives▯rise▯to▯ a▯blue▯indolyl▯compound▯as▯a▯product▯when▯cleaved▯by▯β▯galactosidase.▯▯Most▯ plasmid▯vectors▯have▯a▯portion▯of▯the▯lacZ▯gene▯that▯is▯interrupted▯by▯the▯multiple▯ cloning▯site.▯If▯there▯is▯no▯insert▯in▯the▯vector,▯bacteria▯harboring▯it▯will▯grow▯and▯give▯ rise▯to▯blue▯colonies▯if▯X▯GAL▯is▯added▯to▯the▯plates.▯▯If▯there▯is▯an▯insert▯in▯the▯MCS,▯ this▯generally▯disrupts▯the▯reading▯frame▯of▯the▯lacZ▯gene,▯giving▯rise▯to▯a▯ nonfunctional▯β▯galactosidase.▯▯Thus,▯bacteria▯harboring▯a▯vector▯that▯has▯an▯insert▯ give▯rise▯to▯white▯colonies▯on▯X▯GAL▯containing▯media.▯This▯is▯useful▯when▯trying▯to▯ clone▯something:▯that▯is,▯only▯pursue▯those▯colonies▯that▯are▯white.▯ ▯ o▯nitrophenyl▯b▯D▯galactoside▯(ONPG)▯generates▯a▯yellow▯product▯when▯cleaved▯by▯ β▯galactosidase.▯We▯will▯use▯ONPG▯in▯our▯experiments▯to▯measure▯the▯activity▯of▯the▯ operon.▯▯ ▯ During▯the▯first▯lab▯period,▯you▯will▯use▯wildtype▯or▯mutant▯(lac▯I ▯or▯lac▯Z )▯bacteria▯ that▯have▯been▯grown▯in▯minimal▯media.▯You▯will▯take▯aliquots▯and▯grow▯them▯in▯the▯ presence▯of▯one▯of▯the▯following▯▯ a) glucose▯▯ b) lactose▯ c) sucrose▯ ▯ After▯60▯minutes▯of▯growth,▯you▯will▯measure▯β▯galactosidase▯activity▯by▯the▯ONPG▯ assay,▯using▯the▯Spectronic▯20▯that▯you▯used▯earlier▯in▯the▯course.▯▯ ▯ Each▯bench▯will▯take▯one▯of▯the▯types▯of▯bacteria▯which▯will▯be▯labeled▯A,▯B▯or▯C.▯The▯ genotype▯will▯not▯be▯identified:▯you▯get▯to▯elucidate▯their▯identity!▯You▯will▯initiate▯8▯ cultures▯(so▯that▯there▯will▯be▯duplicates▯tubes▯of▯a▯control▯and▯each▯of▯the▯three▯ sugar▯solutions).▯▯Label▯these▯C=control,▯G=glucose,▯L=lactose,▯and▯S=sucrose.▯▯▯ ▯ ! Lab▯6A:▯in▯this▯lab▯you▯will▯get▯a▯culture▯of▯bacteria▯(genotype▯unknown)▯ which▯you▯will▯grow▯in▯the▯presence▯of▯various▯sugars,▯perform▯the▯ ONGP▯assay▯and▯generate▯graphs▯of▯the▯data.▯Make▯up▯your▯own▯ restriction▯mapping▯problem▯while▯bacteria▯are▯growing.▯ ! Lab▯6B:▯dry▯lab:▯you▯will▯analyze▯results▯of▯lab▯6A▯and▯plan▯your▯own▯ experiment▯for▯lab▯7A▯▯▯ ! Lab▯7A:▯student▯lab▯experiments▯ ! Lab▯7B:▯Analysis▯of▯data▯from▯7A▯&▯re▯design▯of▯your▯experiment▯(as▯needed);▯ restriction▯mapping▯test▯ ! Lab▯8A:▯Repeat▯of▯student▯lab▯experiments▯and▯analysis▯of▯results▯ ▯ ▯ ▯ ▯ ▯ ▯ 3 ▯ Lab▯6A:▯Culture▯of▯bacteria▯in▯various▯sugars▯&▯ONPG▯assay▯ ▯ Materials▯needed:▯ Overnight▯cultures▯of▯wildtype,▯lac▯I ,▯and▯lac▯Z ▯bacteria.▯▯▯▯ Vortex▯mixer▯ M9▯medium▯+▯glycerol▯ ▯ ▯ ▯ ▯ ▯ Culture▯tubes▯ 1%▯glucose▯▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ Spectronic▯20▯tubes▯ 1%▯sucrose▯▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ Parafilm▯ 1%▯lactose▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ 1%▯Sarkosyl▯ 0.5%▯ONPG▯in▯phosphate▯buffer▯▯ ▯ ▯ ▯ ▯ 0.25M▯Na CO ▯2 3 Spectronic▯20▯spectrophotometer▯ ▯ ▯ ▯ ▯ Ice▯bucket▯and▯ice▯ Shaking▯water▯bath▯ ▯ ▯ ▯ ▯ ▯ ▯ Sharpie▯marker▯ Graph▯paper▯ ▯ ▯ ▯ ▯ ▯ ▯ ▯ Sterile▯water▯ Test▯tube▯racks▯ ▯ Preparation▯of▯media▯and▯reagents:▯▯ These▯solutions▯will▯be▯prepared▯for▯you.▯▯ ▯ M9+glycerol▯ ▯ 1%▯lactose▯ 1%▯glucose▯ ▯ 0.25M▯Na CO ▯2 3 1%▯sucrose▯ ▯ ▯▯ ▯ Note:▯During▯the▯90▯minute▯wait▯in▯today’s▯lab,▯each▯group▯will▯work▯together▯ to▯generate▯&▯solve▯a▯restriction▯mapping▯problem.▯Refer▯to▯PDF▯#5▯for▯some▯ instructions▯on▯how▯to▯proceed▯and▯troubleshoot▯the▯problem.▯You▯are▯ required▯to▯present▯this▯problem▯(with▯solution)▯to▯your▯TA▯at▯the▯end▯of▯ today’s▯lab,▯so▯work▯together▯efficiently.▯▯ ▯ Procedure:▯▯ Each▯bench▯will▯receive▯an▯overnight▯culture▯of▯one▯of▯the▯three▯types▯of▯bacteria,▯ which▯will▯be▯labeled▯A,▯B,▯or▯C.▯▯▯▯ ▯ 1. Procure▯8▯culture▯tubes.▯▯Label▯2▯of▯each▯as:▯“C”,▯“S”,▯“L”,▯and▯“G”.▯▯These▯ represent▯the▯control,▯sucrose▯added,▯lactose▯added,▯and▯glucose▯added▯ cultures.▯ 2. Use▯a▯sterile▯pipette▯to▯transfer▯0.5ml▯of▯M9+glycerol▯medium▯to▯each▯tube.▯▯ You▯can▯use▯the▯same▯pipette▯for▯all▯of▯them.▯▯▯ 3. Use▯a▯new▯sterile▯pipette▯to▯transfer▯0.8ml▯of▯the▯overnight▯culture▯to▯each▯of▯ the▯tubes.▯Again,▯you▯can▯use▯the▯same▯pipette▯for▯each▯one.▯▯Be▯sure▯to▯put▯the▯ pipette▯in▯the▯appropriate▯wash▯container.▯ 4. Add▯100µl▯of▯the▯appropriate▯sugar▯to▯each▯tube▯(e.g.▯add▯sterile▯water▯to▯the▯ “C”▯tube,▯1%▯glucose▯to▯the▯“G”▯tube,▯etc.)▯▯ 5. Place▯the▯tubes▯in▯the▯37°C▯shaker▯and▯let▯them▯shake▯for▯60▯minutes.▯▯ ▯ 4 ▯ ▯ *****RESTRICTION▯MAPPING▯PROBLEM*****▯ ▯ 6. Once▯your▯incubation▯period▯is▯over,▯place▯your▯cultures▯on▯ice.▯▯ 7. Add▯one▯drop▯of▯1%▯sarkosyl▯=▯100µl▯
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