APh$162$–$Cellular$Decision$Making$Measuring$Gene$Expression$Winter'2010'$Introduction:'If'you'had'to'choose'between'broccoli'and'chocolate,'would'you'eat'both'simultaneously'or'one'at'a'time?'Which'one'would'you'eat'first?'How'and'what'do'you'think'a'mouse'or'a'fly'would'choose?'What'about'a'bacterium?'''According)to)the)famous)Monod’s)experiment1,'E.'coli'can'choose'to'eat'a'certain'sugar'even'when'multiple'types'of'sugar'are'present'in'the'growth'media.'Today,'we'are'going'to'focus'on'one'of'the'most'well'known'examples—the'lac'operon.'''The'lac'operon'controls'the'production'of'three'different'genes'associated'with'the'metabolism'of'lactose.'One'of'these'genes'is'lacZ,'which'encodes'for'the'enzyme'beta‐galactosid ase'which'metabolizes'lactose.'When'the'growth'media'has'glucose'and'lactose'E.'coli'will'not'produce'any'beta‐galactosidase.'It'will'only'produce'it'once'it'has'consumed'all'the'glucose.'This'means'that'the'cell'has'a'way'of'determining'if'there'is'no'glucose'and'if'there'is'lactose'in'the'media.'''In'this'experiment'we'will'explore'the'part'of'the'circuit'that'takes'care'of'detecting'the'presence'or'absence'of'lactose'in'the'media.'Instead'of'lactose,'which'would'get'eventually'eaten'by'the'cells,'we'use'the'inducer'IPTG.''This'small'molecule'int eracts'with'Lac'repressor'in'much'the'same'way'that'the'real'sugar'does'except'that'it'cannot'be'cleaved'by'the'enzyme'beta‐galactosidase,'making'it'a'useful'experimental'substrate.''''''In'the'absence'of'lactose'or'IPTG,'Lac'repressor'binds'to'an'operator'that'overlaps'th e'lac'promoter.'Becau se'of'this'overlap,'RNA'pol ymer ase'cannot'bind'to'DNA'to'initiate'transcription,'resulting'in'repression.'More'interestingly,'there'are'three'operators'in'the'wild'type'lac'operon'with'different'binding'strengths.'Tetramerized'Lac'repressor'can'even'bind'to'two'operators'at'the'same'time,'resulting'in'a'DNA'loop'which'enhances'the'repression'effect.'' '$Fig$1.$Repression$in$t he$lac$operon.'(A)'A'RNA'polymerase'molecule'binds'to'its'promoter'on'the'DNA'to'initiate'transcription.'(B)'When'a'repressor'molecule'binds'to'its'operator,'it'block s'part'of'the'promoter'and'leads'to'repression.'(C)'A'tetramerized'repressor'can'bind'to'two'op erators'at'the'same'time,'thus'forming'a'DNA'lo op'and'enhancing'the'repression'effect.' 1'Jacques'Monod'" The'growth'of'bacterial'cultures"'Annual'Reviews'Microbiology.'1949.'3:371‐394.'A . B. C .However,)no)response)in)biology)is)really)digital.)There)is)no)such)thing)as)something)being)“on”)or)“off”.)You)can)instead)ask)an)analog)question:)to)what)extent)is)something)off?''The$Construct:'In'this'experiment,'we'will'use'two'synthetic'sequences'for'the'lac'operon:'one'with'a'single'operator'O1,'and'another'one'with'two'operators'Oid'(an'operator'with'ideally'strong'binding'strength)'and'O1.'In'both'of'the'sequences,'the'target'lacZ'gene'was'replaced'by'a'YFP'gene'controlled'by'a'promoter'called'lacUV5'(a'mutant'of'the'wild'type'lac$promoter).'''''' ' '$Fig$2.$The$construct.'(A)'Sequen ce'with'a'single'operator'O1.'(B)'Sequence'with'two'ope rators:'Oid'and'O1.'The'length'of'the'spacer'DNA'between't he'two'operators'is'72.5bp.'The'promoter'in'both'of'the'sequences'is'lacUV5'controlling'the'expression'of'a'YFP'gene.''Measuring$gene$expression:'We'will'measure'the'fluorescence'expressed'by'cells'containing'the'construct'described'above'for'different'concentrations'of'IPTG.'We'want'to'define'a'fold‐change'in'gene'expression,'a'measure'of'the'relative'change'in'gene'expression'upon'the'addition'of'inducer.'''One'problem'with'measuring'fluorescence'in'cells'is'that'the'cells'are'fluorescent'themselves!'This'means'that'if'you'measure'the'fluorescence'of'a'cell'expressing'YFP'you'wi l l 'actually'be'measuring'the'fluorescence'of'the'YFP'molecules'on'top'of'the'autofluorescence'of'the'cell.'In'order'to'account'for'this'you'can'measure'the'autofluorescence'of'a'cell'that'lacks'any'YFP.'''We'will'define'the'fold‐change'in'gene'expression'as''''Here,'AutoFluo'is'the'autofluorescence'and'Fluo([LacI])'is'the'fluorescence'in'cells'with'varied'amounts)of)Lac)repressors.)However,)since)we)can’t)directly)measure)the)concentration)of)Lac)repressors,'but'we'can'control'the'amount'of'IPTG,'instead'we'can'express'the'fold‐change'as''''To)measure)Fluo([LacI≠0]),'we'use'a'modified'strain'of'cells' with'the'same'YFP'gene'but'no'Lac'repressor.''A . B.Note'that'Fluo[IPTG]'refers'to'the'total'integrated'fluorescence'in'a' cell.'This'is'because'we'are'interested'in'the'total'gene'expression'in'a'cell.'How'will'you'correct'for'autofluorescence,'given'that'cells'have'different'areas'(i.e.,'how'should'you'calculate'AutoFluo)?''In'order'to'quantify'the'level'of'gene'expression'you'will'need'to'write'code'in'MatLab'or'other'language'of'your'choice.'You'will 'have'to'find'the'cells'au tomatically'and'use'the'mask'in'order'to'integrate'over'the'fluorescence'of'each'cell.'''Experimental$protocol:'What'happened'behind'the'scenes:''Five'hours'before'you'started'your'experiment'we'diluted'cells'from'an'LB'overnight'culture'into'minimal'media'in'the'presence'of'different'concentrations'of'IPTG.'This'particular'minimal'media'has'salts,'a'carbon'source'and'some'amino'acids.''''We'also'prepared'agar'pads'now'made'with'PBS'(phosphate'buffer).'Cell'cannot'grow'in'this'medium.'The'idea'is'that'we'will'be'taking'snapshots'of'their'state'when'we'took'them'out'of'the'culture.'''Due'to'time'limitation,'we'had'mounted'the'samples'on'the'Wilco'dishes'for'yo u.'For'each'construct,'there'are'8'samples:'0uM'(no'IPTG'in'the'media),'10uM,'50uM,'100uM,'500uM,'1mM,'NoLacI,'and'NoFluo.'Thus,'you'will'have'two'Wilco'dishes'and'8'agar'pads'on'each'plate.'Before'starting'imaging,'we'had'let'the'pads'equilibrate'to'the'temperatu
View Full Document