Bacterial Transformation Lab Report.pdf - Bacterial...

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Unformatted text preview: Bacterial​ ​Transformation Abstract: The​ ​objective​ ​of​ ​this​ ​experiment​ ​is​ ​to​ ​conduct​ ​bacterial​ ​transformation​ ​on​ ​E.​ ​coli​ ​bacteria,​ ​by successfully​ ​inserting​ ​a​ ​plasmid,​ ​pGLO,​ ​and​ ​getting​ ​the​ ​bacteria​ ​to​ ​express​ ​a​ ​gene​ ​that​ ​allows​ ​it to​ ​glow​ ​under​ ​UV​ ​light.​ ​This​ ​gene​ ​is​ ​called​ ​the​ ​GFP​ ​gene.​ ​The​ ​GFP​ ​gene​ ​not​ ​only​ ​allows​ ​the bacteria​ ​to​ ​grow,​ ​but​ ​it​ ​also​ ​gives​ ​the​ ​bacteria​ ​a​ ​resistance​ ​to​ ​ampicillin.​ ​If​ ​this​ ​experiment​ ​were to​ ​have​ ​been​ ​conducted​ ​with​ ​no​ ​errors​ ​whatsoever,​ ​the​ ​majority​ ​of​ ​the​ ​bacterial​ ​colonies​ ​should have​ ​been​ ​able​ ​to​ ​grow​ ​and​ ​glow.​ ​Unfortunately,​ ​in​ ​this​ ​experiment​ ​that​ ​was​ ​not​ ​the​ ​case.​ ​While none​ ​of​ ​the​ ​colonies​ ​glowed,​ ​some​ ​were​ ​still​ ​able​ ​to​ ​grow,​ ​due​ ​to​ ​the​ ​expression​ ​of​ ​the​ ​bla​ ​gene. Although​ ​the​ ​hypothesis​ ​was​ ​not​ ​supported,​ ​it​ ​was​ ​most​ ​likely​ ​a​ ​flaw​ ​in​ ​human​ ​error​ ​and​ ​not​ ​in the​ ​experiment​ ​itself. Introduction: Genes​ ​are​ ​codes​ ​inside​ ​of​ ​DNA​ ​that​ ​dictate​ ​which​ ​proteins​ ​are​ ​made​ ​within​ ​an​ ​organism.​ ​These tiny​ ​chunks​ ​of​ ​DNA​ ​are​ ​essentially​ ​instructions​ ​that​ ​make​ ​up​ ​living​ ​organisms.​ ​Similar​ ​to evolution,​ ​transformation​ ​occurs​ ​within​ ​nature​ ​when​ ​a​ ​certain​ ​trait​ ​becomes​ ​favorable​ ​and​ ​useful for​ ​an​ ​organism.​ ​Genetic​ ​transformation​ ​occurs​ ​when​ ​a​ ​gene​ ​is​ ​inserted​ ​into​ ​an​ ​organism​ ​in​ ​order to​ ​change​ ​a​ ​certain​ ​trait​ ​of​ ​that​ ​organism​ ​for​ ​the​ ​organism’s​ ​own​ ​benefit.​ ​In​ ​this​ ​experiment,​ ​a plasmid,​ ​pGLO,​ ​will​ ​be​ ​inserted​ ​into​ ​the​ ​genome​ ​of​ ​E.​ ​coli​ ​bacteria​ ​by​ ​the​ ​process​ ​of​ ​heat​ ​shock. This​ ​will​ ​be​ ​done​ ​so​ ​that​ ​the​ ​GFP​ ​gene​ ​will​ ​be​ ​produced,​ ​giving​ ​the​ ​bacteria​ ​the​ ​ability​ ​to​ ​glow under​ ​UV​ ​light​ ​as​ ​well​ ​as​ ​resistance​ ​to​ ​an​ ​antibiotic—ampicillin.​ ​The​ ​production​ ​of​ ​GFP​ ​also depends​ ​on​ ​two​ ​things:​ ​the​ ​presence​ ​of​ ​arabinose​ ​sugar​ ​and​ ​the​ ​araC​ ​gene.​ ​Not​ ​all​ ​of​ ​the​ ​petri dishes​ ​will​ ​include​ ​the​ ​pGLO​ ​plasmid.​ ​In​ ​those​ ​that​ ​do,​ ​the​ ​bla​ ​gene​ ​will​ ​produce​ ​the​ ​bla​ ​protein. First,​ ​the​ ​bla​ ​gene​ ​will​ ​be​ ​transcribed​ ​by​ ​the​ ​bla​ ​mRNA​ ​and​ ​then​ ​it​ ​will​ ​be​ ​translated​ ​into​ ​the​ ​bla protein.​ ​The​ ​bla​ ​protein’s​ ​purpose​ ​is​ ​to​ ​give​ ​the​ ​E.​ ​coli​ ​bacteria​ ​resistance​ ​to​ ​ampicillin.​ ​The presence​ ​of​ ​the​ ​bla​ ​gene​ ​will​ ​enable​ ​growth​ ​in​ ​the​ ​bacteria​ ​in​ ​some​ ​cultures,​ ​but​ ​not ​in​ ​others. The​ ​other​ ​cultures​ ​that​ ​the​ ​E.​ ​coli​ ​bacteria​ ​will​ ​not​ ​be​ ​able​ ​to​ ​grow​ ​in​ ​will​ ​be​ ​those​ ​lacking​ ​the presence​ ​of​ ​the​ ​bla​ ​gene.​ ​The​ ​same​ ​thing​ ​that​ ​happened​ ​to​ ​the​ ​bla​ ​gene​ ​will​ ​also​ ​happen​ ​to​ ​the araC​ ​gene.​ ​The​ ​araC​ ​gene​ ​will​ ​first​ ​be​ ​transcribed​ ​by​ ​the​ ​araC​ ​mRNA​ ​and​ ​then​ ​translate​ ​into​ ​the araC​ ​protein.​ ​The​ ​araC​ ​protein​ ​is​ ​able​ ​to​ ​either​ ​positively​ ​or​ ​negatively​ ​regulate​ ​the​ ​expression​ ​of proteins​ ​and,​ ​in​ ​this​ ​case,​ ​the​ ​protein​ ​that​ ​is​ ​being​ ​regulated​ ​for​ ​this​ ​experiment​ ​is​ ​the​ ​GFP protein.​ ​The​ ​relationship​ ​between​ ​the​ ​araC​ ​protein​ ​and​ ​the​ ​GFP​ ​protein​ ​is​ ​directly​ ​related.​ ​In​ ​the presence​ ​of​ ​the​ ​araC​ ​protein​ ​the​ ​GFP​ ​protein​ ​is​ ​expressed,​ ​while​ ​in​ ​the​ ​absence​ ​of​ ​the​ ​GFP protein​ ​it​ ​is​ ​not​ ​expressed.​ ​In​ ​theory,​ ​without​ ​any​ ​experimental​ ​errors,​ ​the​ ​samples​ ​expressing both​ ​the​ ​GFP​ ​gene​ ​and​ ​the​ ​araC​ ​protein​ ​will​ ​express​ ​the​ ​bla​ ​protein,​ ​giving​ ​the​ ​bacteria resistance​ ​to​ ​ampicillin,​ ​as​ ​well​ ​as​ ​allow​ ​it​ ​to​ ​glow​ ​under​ ​UV​ ​light.​ ​The​ ​samples​ ​that​ ​do​ ​not express​ ​the​ ​bla​ ​protein​ ​will​ ​die​ ​in​ ​the​ ​presence​ ​of​ ​ampicillin,​ ​hence​ ​no​ ​growth​ ​or​ ​glow​ ​will​ ​occur. Materials​ ​and​ ​Methods: First,​ ​two​ ​micro​ ​test​ ​tubes​ ​will​ ​be​ ​needed.​ ​One​ ​of​ ​the​ ​test​ ​tubes​ ​will​ ​be​ ​used​ ​for​ ​those​ ​petri​ ​dishes with​ ​the​ ​pGLO​ ​included​ ​in​ ​it​ ​and​ ​the​ ​other​ ​one​ ​will​ ​be​ ​for​ ​those​ ​petri​ ​dishes​ ​without​ ​the​ ​pGLO​ ​in it.​ ​Make​ ​sure​ ​to​ ​label​ ​these​ ​so​ ​that​ ​the​ ​test​ ​tubes​ ​do​ ​not​ ​get​ ​mixed​ ​up—separation​ ​of​ ​the​ ​pGLO samples​ ​is​ ​necessary​ ​for​ ​this​ ​experiment.​ ​Place​ ​the​ ​two​ ​micro​ ​test​ ​tubes​ ​into​ ​a​ ​foam​ ​tube​ ​rack and​ ​use​ ​a​ ​pipette​ ​to​ ​fill​ ​each​ ​of​ ​them​ ​with​ ​250​ ​uL​ ​(microliters)​ ​of​ ​transformation​ ​solution​ ​and close​ ​both​ ​of​ ​the​ ​lids​ ​on​ ​each​ ​of​ ​the​ ​micro​ ​test​ ​tubes.​ ​Then,​ ​take​ ​the​ ​foam​ ​rack​ ​with​ ​the​ ​tubes​ ​in​ ​it and​ ​place​ ​it​ ​in​ ​a​ ​covered​ ​ice​ ​bucket​ ​for​ ​three​ ​minutes.​ ​After​ ​three​ ​minutes,​ ​remove​ ​the​ ​foam​ ​rack from​ ​the​ ​ice​ ​and​ ​open​ ​a​ ​new,​ ​sterile​ ​loop​ ​from​ ​its​ ​package​ ​and​ ​remove​ ​it​ ​from​ ​the​ ​opposite​ ​end of​ ​where​ ​the​ ​loop​ ​is​ ​located.​ ​Use​ ​the​ ​loop​ ​to​ ​pick​ ​up​ ​a​ ​single​ ​colony​ ​of​ ​E.​ ​coli,​ ​open​ ​one​ ​of​ ​the micro​ ​test​ ​tubes’​ ​lid ​and​ ​submerge​ ​the​ ​loop​ ​into​ ​the​ ​transformation​ ​solution.​ ​Using​ ​a​ ​new,​ ​sterile loop​ ​for​ ​each​ ​trial,​ ​repeat​ ​this​ ​step​ ​for​ ​both​ ​of​ ​the​ ​micro​ ​test​ ​tubes,​ ​the​ ​-pGLO​ ​and​ ​the​ ​+pGLO. After​ ​both​ ​of​ ​the​ ​micro​ ​test​ ​tubes​ ​have​ ​been​ ​contaminated​ ​with​ ​the​ ​E.​ ​coli​ ​place​ ​them​ ​back​ ​into the​ ​covered​ ​ice​ ​bucket​ ​for​ ​another​ ​three​ ​minutes.​ ​This​ ​time,​ ​after​ ​removing​ ​the​ ​foam​ ​tray​ ​from the​ ​ice​ ​bucket,​ ​only​ ​open​ ​the​ ​the​ ​+pGLO​ ​tube—leave​ ​the​ ​-pGLO​ ​tube​ ​closed.​ ​Grab​ ​another​ ​new, sterile​ ​loop​ ​and​ ​insert​ ​it​ ​into​ ​the​ ​pGLO​ ​plasmid​ ​DNA​ ​stock​ ​tube.​ ​Stir​ ​the​ ​loop​ ​in​ ​the​ ​solution​ ​and when​ ​it​ ​is​ ​removed​ ​there​ ​should​ ​be​ ​a​ ​film​ ​over​ ​the​ ​loop,​ ​which​ ​indicates​ ​that​ ​the​ ​DNA​ ​has​ ​been picked​ ​up​ ​by​ ​the​ ​loop.​ ​Stick​ ​the​ ​loop​ ​into​ ​the​ ​+pGLO​ ​tube​ ​ONLY​ ​and​ ​stir.​ ​Remove​ ​the​ ​loop​ ​and close​ ​the​ ​lid​ ​on​ ​the​ ​+pGLO​ ​once​ ​again,​ ​and​ ​place​ ​the​ ​foam​ ​tray​ ​with​ ​the​ ​tubes​ ​back​ ​into​ ​the​ ​ice. This​ ​time,​ ​make​ ​sure​ ​that​ ​the​ ​tubes​ ​are​ ​pushed​ ​all​ ​the​ ​way​ ​down​ ​in​ ​the​ ​foam​ ​rack​ ​and​ ​that​ ​the tubes​ ​are​ ​fully​ ​submerged​ ​in​ ​the​ ​ice.​ ​The​ ​tubes​ ​will​ ​sit​ ​in​ ​the​ ​ice​ ​for​ ​ten​ ​minutes.​ ​While​ ​waiting for​ ​the​ ​tubes​ ​to​ ​be​ ​done​ ​in​ ​the​ ​ice​ ​bucket,​ ​acquire​ ​the​ ​following​ ​plates:​ ​1​ ​LB​ ​plate​ ​(no​ ​lines labeled​ ​on​ ​it),​ ​2​ ​LB/AMP​ ​plates​ ​(1​ ​black​ ​line​ ​labeled​ ​on​ ​it),​ ​1​ ​LB/araC​ ​(2​ ​black​ ​lines​ ​labeled​ ​on it)​ ​and​ ​1​ ​LB/AMP/araC​ ​(1​ ​red​ ​line​ ​labeled​ ​on​ ​it).​ ​On​ ​the​ ​bottom,​ ​label​ ​each​ ​of​ ​these​ ​plates​ ​with your​ ​group’s​ ​name​ ​or​ ​number,​ ​the​ ​type​ ​of​ ​plate​ ​that​ ​it​ ​is,​ ​your​ ​lab​ ​TA’s​ ​name,​ ​and​ ​whether​ ​or​ ​not the​ ​pGLO​ ​plasmid​ ​was​ ​exposed​ ​to​ ​the​ ​E.​ ​coli​ ​sample.​ ​After​ ​the​ ​ten​ ​minutes​ ​is​ ​up,​ ​remove​ ​the foam​ ​tray​ ​and​ ​place​ ​it​ ​into​ ​a​ ​water​ ​bath,​ ​set​ ​at​ ​42​ ​degrees​ ​celsius,​ ​for​ ​50​ ​seconds.​ ​After​ ​the​ ​50 seconds​ ​is​ ​up,​ ​immediately​ ​transfer​ ​the​ ​foam​ ​tray​ ​directly​ ​back​ ​into​ ​the​ ​ice​ ​bucket.​ ​Immediate transfer​ ​allows​ ​for​ ​optimum​ ​transformation​ ​results​ ​to​ ​occur.​ ​Let​ ​the​ ​tubes​ ​sit​ ​in​ ​the​ ​ice​ ​bucket​ ​for another​ ​two​ ​minutes.​ ​Remove​ ​the​ ​foam​ ​tray​ ​with​ ​the​ ​tubes​ ​from​ ​the​ ​ice​ ​bucket​ ​for​ ​the​ ​last​ ​time and​ ​place​ ​the​ ​tray​ ​on​ ​the​ ​laboratory​ ​table.​ ​Open​ ​one​ ​of​ ​the​ ​tubes,​ ​grab​ ​a​ ​new,​ ​sterile​ ​pipette, extract​ ​250​ ​uL​ ​of​ ​LB​ ​nutrient​ ​broth​ ​and​ ​put​ ​it​ ​into​ ​the​ ​tube.​ ​Make​ ​sure​ ​to​ ​mix​ ​the​ ​LB​ ​nutrient well​ ​with​ ​the​ ​tube’s​ ​solution​ ​and​ ​then​ ​close​ ​the​ ​lid.​ ​Using​ ​another​ ​new,​ ​sterile​ ​pipette,​ ​repeat​ ​this step​ ​again​ ​for​ ​the​ ​other​ ​tube.​ ​After​ ​both​ ​tubes​ ​have​ ​received​ ​the​ ​LB​ ​nutrient,​ ​let​ ​the​ ​tubes​ ​sit​ ​at room​ ​temperature​ ​for​ ​20​ ​minutes.​ ​ ​Tap​ ​each​ ​of​ ​the​ ​closed​ ​tubes​ ​to​ ​mix​ ​the​ ​solution.​ ​Use​ ​a​ ​new, sterile​ ​pipette​ ​and​ ​extract​ ​exactly​ ​100​ ​uL​ ​of​ ​solution​ ​from​ ​one​ ​of​ ​the​ ​tubes​ ​and​ ​put​ ​it​ ​onto​ ​the corresponding​ ​petri​ ​dish.​ ​Repeat​ ​this​ ​step​ ​for​ ​both​ ​of​ ​the​ ​tubes,​ ​using​ ​a​ ​total​ ​of​ ​two​ ​new,​ ​sterile pipettes​ ​(one​ ​for​ ​each).​ ​For​ ​each​ ​of​ ​the​ ​plates,​ ​use​ ​a​ ​new,​ ​sterile​ ​loop​ ​and​ ​spread​ ​the​ ​suspensions evenly​ ​around​ ​the​ ​surface.​ ​Be​ ​sure​ ​to​ ​not​ ​press​ ​the​ ​loop​ ​too​ ​deep​ ​into​ ​the​ ​jar—just​ ​lightly​ ​brush across​ ​the​ ​surface​ ​with​ ​the​ ​loop.​ ​Stack​ ​the​ ​plates​ ​in​ ​order,​ ​-pGLO/LB​ ​being​ ​on​ ​the​ ​bottom​ ​and +pGLO/LB+AMP+ara​ ​being​ ​on​ ​the​ ​top,​ ​and​ ​tape​ ​them​ ​together​ ​along​ ​the​ ​sides​ ​of​ ​the​ ​plates. Label​ ​the​ ​bottom​ ​of​ ​the​ ​stack​ ​with​ ​the​ ​appropriate​ ​group​ ​name/number​ ​and​ ​class​ ​period.​ ​Place the​ ​stack​ ​upside​ ​down​ ​inside​ ​of​ ​the​ ​incubator.​ ​Check​ ​back​ ​for​ ​results​ ​after​ ​one​ ​day.​ ​The independent​ ​variable​ ​in​ ​this​ ​experiment​ ​was​ ​the​ ​presence​ ​or​ ​the​ ​lack​ ​of​ ​the​ ​plasmid​ ​(pGLO).​ ​The dependent​ ​variable​ ​in​ ​this​ ​experiment​ ​was​ ​the​ ​bacteria​ ​growth​ ​and​ ​the​ ​fluorescence​ ​of​ ​the bacteria.​ ​The​ ​controls​ ​of​ ​the​ ​experiment​ ​were​ ​the​ ​sterile​ ​pipette,​ ​sterile​ ​loop,​ ​the​ ​ice​ ​bucket​ ​and the​ ​water​ ​bath. The​ ​second​ ​row​ ​of​ ​petri​ ​dishes​ ​is​ ​a​ ​group​ ​that​ ​was​ ​successful​ ​in​ ​getting​ ​the​ ​GFP​ ​gene​ ​to​ ​be expressed.​ ​The​ ​third​ ​row​ ​is​ ​the​ ​row​ ​of​ ​results​ ​for​ ​this​ ​experiment. Results: E.​ ​Coli​ ​Growth​ ​and​ ​Fluorescence -pGLO LB -pGLO LB​ ​+​ ​AMP +pGLO LB​ ​+​ ​AMP +pGLO LB​ ​+​ ​ara (1) (2) (3) (4) +pGLO LB​ ​+​ ​AMP +ara (5) Growth Present (+​ ​if​ ​present and​ ​-​ ​if​ ​not present) + + - + - Fluorescent Under​ ​UV Light (Actual) No​ ​Glow No​ ​Glow No​ ​Glow No​ ​Glow No​ ​Glow Fluorescent Under​ ​UV Light (Prediction) No​ ​Glow No​ ​Glow Glow Glow Glow Discussion: For​ ​the​ ​Bacterial​ ​Transformation​ ​experiment,​ ​the​ ​hypothesis​ ​was​ ​neither​ ​supported​ ​or​ ​rejected. Although​ ​colonies​ ​were​ ​formed,​ ​the​ ​samples​ ​did​ ​not​ ​glow​ ​and​ ​the​ ​GFP​ ​gene​ ​was​ ​not​ ​expressed in​ ​any​ ​of​ ​the​ ​colonies.​ ​Those​ ​colonies​ ​expressing​ ​the​ ​bla​ ​protein​ ​promoted​ ​growth,​ ​while​ ​those repressing​ ​the​ ​bla​ ​protein​ ​died​ ​in​ ​the​ ​presence​ ​of​ ​ampicillin,​ ​and​ ​this​ ​held​ ​true​ ​for​ ​the experiment.​ ​The​ ​expression​ ​of​ ​the​ ​bla​ ​protein,​ ​along​ ​with​ ​the​ ​presence​ ​of​ ​the​ ​ampicillin,​ ​only partially​ ​supports​ ​the​ ​hypothesis.​ ​The​ ​samples​ ​that​ ​had​ ​the​ ​plasmid​ ​inserted​ ​into​ ​it​ ​did​ ​not support​ ​the​ ​hypothesis​ ​since​ ​none​ ​of​ ​the​ ​cultures​ ​expressed​ ​the​ ​glowing​ ​trait.​ ​ ​Possible​ ​causes​ ​for the​ ​GFP​ ​gene​ ​being​ ​repressed​ ​might​ ​be​ ​because​ ​of​ ​a​ ​lack​ ​of​ ​plasma​ ​being​ ​inserted​ ​into​ ​the sample​ ​(not​ ​enough)​ ​or​ ​from​ ​the​ ​timing​ ​being​ ​off​ ​enough​ ​to​ ​the​ ​point​ ​where​ ​the​ ​plasma​ ​was​ ​not weak​ ​enough​ ​to​ ​get​ ​into​ ​the​ ​E.​ ​coli.​ ​The​ ​transfer​ ​from​ ​the​ ​water​ ​bath​ ​to​ ​the​ ​ice​ ​could​ ​have​ ​been too​ ​slow​ ​to​ ​properly​ ​allow​ ​transformation.​ ​In​ ​any​ ​experiment,​ ​with​ ​human​ ​regulation,​ ​there​ ​is always​ ​room​ ​for​ ​human​ ​error​ ​and​ ​that​ ​is​ ​what​ ​I​ ​believe​ ​caused​ ​the​ ​flaws​ ​in​ ​this​ ​experiment—I​ ​do not​ ​believe​ ​it​ ​was​ ​a​ ​flaw​ ​within​ ​the​ ​experimental​ ​design​ ​itself.​ ​A​ ​future​ ​implication​ ​of​ ​this experiment​ ​could​ ​eventually​ ​lead​ ​to​ ​the​ ​improvement​ ​of​ ​antibiotics,​ ​how​ ​to​ ​make​ ​disease/illness glow​ ​within​ ​living​ ​organisms​ ​so​ ​that​ ​they​ ​can​ ​be​ ​targeted​ ​and​ ​cured​ ​in​ ​an​ ​appropriate​ ​manner, and​ ​also​ ​to​ ​help​ ​scientists​ ​further​ ​understand​ ​the​ ​manifestation​ ​process​ ​of​ ​E.​ ​coli​ ​bacteria​ ​and how​ ​to​ ​contain​ ​it. ...
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