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New and Improved: The Art of Bacterial Transformation

Introduction

Purpose: The objectives of the New and Improved: Bacterial CultureTransformation Lab are: to observe standard bacterial growth under variousconditions including the transformation of bacteria to understand how the processof transformation occurs! as well as the biological results and conse"uences that

come of transformation and to understand the importance of transformation inpro#ar$otic and eu#ar$otic life c$cles%

Bac#ground: Transformation is the &process b$ which the genetic materialcarried b$ an individual cell is altered b$ the incorporation of foreign'e(ogenous) *NA into its genome+ ',edicineNet%com! &*efinition of -enetictransformation+)% Transformation in bacterial cells occurs when the cellincorporates na#ed *NA into its genetic material in a laborator$ setting! this isencouraged b$ placing the mi(tures of transformation solution and plasmid *NA 'in.p-L/ tube onl$) on ice! then rapidl$ transferring them to a hot water bath forabout fift$ seconds! and then placing them bac# on ice again 0 this procedure iscalled heat shoc# and &increases the permeabilit$ of the cell membrane to *NA+'lab directions)% The agent which the new genetic material is incorporated into

is the bacterial plasmid%A plasmid is a circular deo($ribonucleic acid '*NA) molecule that replicates

independentl$ of the bacterial chromosome and often is the avenue for which abacteria gains resistance to an antibiotic% 1ecombinant plasmids are those whichhave *NA from two or more sources incorporated into a single plasmid% To ma#erecombinant plasmids! two different plasmids are cut with the same restrictionen2$me: this restriction en2$me onl$ cuts at particular restriction sites! so thet$pe of cut it ma#es in one plasmid will be the same t$pe of cut in anotherplasmid% The cut must produce &stic#$ ends+ so that the plasmid *NA can bind toan$ other plasmid *NA with complementar$ base pairs% /nce cut! the two plasmidsare mi(ed and the complementar$ stic#$ ends for each plasmid are sealed b$ *NALigase%The p-L/ plasmid 0 which contains the genes for -3P! or -reen 3luorescent Protein!

and the en2$me 45lactamase! which provides resistance to the antibiotic ampicillin0 will be incorporated into the genome of the 6% Coli bacteria used in the lab%p-L/ is originall$ &from the bioluminescent jell$fish! Ae"uorea 7ictoria whichallows the jell$fish to fluoresce and glow in the dar#% 6% coli can betransformed to+ ma#e the -3P protein and &e(press this gene+ which will &cause the6% coli to glow green when e(posed to 87 light+ 'The ,$ster$ Behind p-lo and -3P)%

The aforementioned antibiotic ampicillin is used in this lab to demonstratethe effect of recombinant plasmids and transformation% B$ appl$ing ampicillin toa Petri dish with and without p-L/! we can see the success of an antibioticagainst bacteria as well as the success of recombinant bacteria against anantibiotic% 9ince p-L/ harbors 45lactamase 0 a restriction en2$me which cuts the*NA of an antibiotic to render it ineffective 0 along with -3P! an$ bacteria withthe p-L/ recombinant plasmid can better resist the antibiotic ampicillin%

In our e(periment! the control treatment is the dish with bacteria! butlac#ing p-L/! ampicillin and agarose% /n this dish! we can see how the bacteriagrow unimpeded or aided b$ an$ other substance% The e(perimental groups are theplate with bacteria! ampicilline and p-L/! the plate with onl$ bacteria andampicillin! and the plate with ampicillin! agarose and p-L/%

$pothesis:;hat plates will have growth and wh$

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recombinant plasmid%;hat plates will glow under a 87 light and wh$

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therefore become resistant to the antibiotic% The plate with p-L/! agarose andampicillin grew nearl$ one5and5a5half times the number of colonies as the platewith onl$ p-L/ and ampicillin because the sugar agarose is a nutrient for thebacteria which helps them divide at a "uic#er rate% The onl$ plate to glow wasthe plate with ampicillin! agarose and p-L/ a possible e(planation for wh$ thisplate glowed when e(posed to 87 light but the plate with onl$ p-L/ and ampicillindid not is that the presence of agarose causes the operon that is responsible forglowing to become activated in order to brea# down agarose and ma#e it usable b$

the bacterial cell%;e #now transformation occurred because there would be no bacterial colonies

on the plate with both p-L/ and ampicillin if transformation had not occurred%The plasmids on this plate had to be recombinant in order to survive in thepresence of an antibiotic% The transformation efficienc$ of DF?%? indicatesthat appro(imatel$ DFG cells were transformed for ever$ one microgram of *NA%

Possible sources of error in our e(periment include inaccuratel$ measuringthe number of bacterial colonies that grew on each plate! insufficientl$ mi(ingthe solutions of *NA after adding restriction en2$mes or not returning themi(tures to ice "uic#l$ enough after during the heat shoc#%

Literature Cited

H*efinition of -enetic Transformation%H ,edicineNet% E June DKKK% D? Apr% ?EEhttp:>>www%medterms%com>script>main>art%asp>department%monm%edu>chemistr$>chemistr$GGE>fall?EE?>nauclair>%