SalemBelay

Mrs.Peterson
APBiology
February1st2015
TransformationLabWriteUp

Introduction:
BacterialtransformationisaprocessinwhichforeignDNAfromaplasmidcanbe
transferredintobacteria(E.Coliinthisexperiment),therebytransformingandaltering
thestateoftheoriginalbacterialcell.Inthisexperiment,aplasmidcalledpGLOwas
used.Transformationbeginsattheoriginofreplication,wherearestrictionenzymewill
cutaroundthegeneofinterestintheplasmid,andthatsamerestrictionenzymewillcut
aroundthebacteriasothatthestickyendsofthetwosegmentswilljointogethertocreate
atransformedbacterialcell.
Aplasmidisasmall,circularportionofDNAthatisphysicallyseparatefrom
chromosomalDNAandisabletoreplicateonitsown.Whenaddedtodifferentbacteria,
plasmidsusuallygivebacteriatheabilitytodovarioustasksdependingontheirfunctions
(e.g.fertilityplasmidsenablereproduction,andresistanceplasmidsthatgivebacteria
antibioticresistance).ThepGLOusedinthisexperimentcontainedampicillinresistance
(anantibiotic),glowingfluorescenceprotein(GFP)fromjellyfish,andAraC,andwas
selectivelyusedthroughouttheexperiment.
Theampicillin/antibioticresistancepresentintheplasmidallowsforbacterialgrowth
despitethepresenceofampicillin.Withnoresistancetoampicillin,asintrialfour(
pGLOwithLB/Amp)alloftheE.Colishouldbekilledandthereshouldbenofurther
growth.
ApromoterisaregionofDNAthatsignalsthebeginningoftranscription.Inthis
experiment,thepromoterispBAD.Ifarabinoseispresentinasolution,thepromoter
pBADwillthenbindthearabinosewiththeAraCfromtheplasmidandpromoteRNA
polymerasebinding.ThiswillleadtothetranscriptionofGFPmRNAandcreatemore
GFPproteins.Thisisseenintrialtwo.Therefore,theGFPproteininthisexperimentis
regulatedbythepresence/absenceofarabinose(Ara)thismeansthattheGFPprotein,
whichisresponsiblefortheglowoftheE.Coli,canonlyworkefficientlyandallow

bacterialglowingwhenitisactivatedbythepresenceofanarabinosesugar(Ara)asseen
intrialtwo.
Results:
Trial
+pGLO
LB/Amp

Data,Observations,andResultsfortheFourTrials
Picture
GeneralGrowth Color/Glow
Somecolonies,
Clearish
apparentgrowth yellow/noglow

+pGLO
LB/Amp/Ara

Lotsofbig
colonies

Clearish
yellow/glow
present

pGLO
LB

Lotsofbig
colonies

Clearish
yellow/noglow

pGLO
LB/Amp

Somepresence
ofcolonies,
apparentgrowth

Clearish
yellow/noglow

Other

Shouldnothave
grownbecause
Amp,an
antibioticwas
present,butitdid
growbecauseof
experimental
errors

Conclusion:
1. Was transformation successful? How do you know? Explain.
Theresults

provethattransformationwassuccessfulinthis

experiment.
Trialsone

andtwowere+pGLO(pGLOwaspresent),soDNA

fromthe

plasmidwaspresentintheE.Coli.Trialonehad

LB/Amp,

andthereforehadsomegrowth,assomecellswere

abletopick

uptheampicillinresistancefromtheplasmid,and

were

thereforeabletoreproduce.TrialtwohadLB/Amp/Ara

andwasthe

onlytrialthatdisplayedbothgrowthandglowasit

contained

ampicillinresistancethatenabledgrowthandthe

arabinose

sugar(Ara)thatenabledaglow.

Trialsthree

andfourwerepGLO(pGLOwasnotpresent),sothe

bacterial

cellshadnointeractionwiththeplasmid.Trialthree

only

containedLB(noampicillintokilloffthebacteria),

and

thereforeunderwentlotsofgrowth.Trialfour

contained

LB/Ampwithnoantibioticresistancebecauseitwas

pGLO,so

itshouldhaveshownnogrowth.However,duetoearly
experimentalerrorsinoverheatingtheplates,the

ampicillin

wasdenaturedandthereforeineffectiveinstoppingthe

bacterial

growth.

2.

How would you improve the experiment to get better

results next time?

Overheating the plates (specifically the pGLO with LB/Amp) was the experimental
error in this lab. To avoid delicate errors like these, one can not only be careful and

precise in every step of the lab, but also repeat the trials several times to ensure that the
results received are most accurate.
3. What advantage would there be for an organism to be able to turn on or off
particular genes in response to certain conditions?
There are many advantages to gene regulation. One advantage could simply be
conserving energy and resources by avoiding overproduction, as seen in the lac operon.
It would be wasteful for an organism to continue producing lactase if there is no lactose
present, so the operon turns off the genes that are necessary to produce lactase when there
is no lactose available. Conversely, if there is lactose present, the production of lactase is
not wasteful and serves a purpose for the organism.
4. What are some potential applications of transforming bacteria?
Astechnologyimproves,theusesforgeneticengineeringareexpandingvastly.
TransformationcouldbeusedinDNAcloning,inthecreationgeneticallymodified
foods,andinmedicaltreatmentslikecreatinglargeamountsofspecificproteinssuchas
insulin,whichhelpstreathumanswithdiabetes.Transformationopensupmanynew
doorsinthedevelopmentofscienceandwithtime,itcanhelpleadtothediscoveryof
newandimprovedmedicinesandtechnologies.