New and Improved: The Art of Bacterial Transformation Introduction Purpose: The objectives of the New and Improved:

Bacterial Culture Transformation Lab are: to observe standard bacterial growth under various conditions including the transformation of bacteria to understand how the process of transformation occurs! as well as the biological results and conse"uences that come of transformation and to understand the importance of transformation in pro#ar$otic and eu#ar$otic life c$cles% Bac#ground: Transformation is the &process b$ which the genetic material carried b$ an individual cell is altered b$ the incorporation of foreign 'e(ogenous) *NA into its genome+ ',edicineNet%com! &*efinition of -enetic transformation+)% Transformation in bacterial cells occurs when the cell incorporates na#ed *NA into its genetic material in a laborator$ setting! this is encouraged 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 for about fift$ seconds! and then placing them bac# on ice again 0 this procedure is called 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 a bacteria gains resistance to an antibiotic% 1ecombinant plasmids are those which have *NA from two or more sources incorporated into a single plasmid% To ma#e recombinant plasmids! two different plasmids are cut with the same restriction en2$me: this restriction en2$me onl$ cuts at particular restriction sites! so the t$pe of cut it ma#es in one plasmid will be the same t$pe of cut in another plasmid% The cut must produce &stic#$ ends+ so that the plasmid *NA can bind to an$ other plasmid *NA with complementar$ base pairs% /nce cut! the two plasmids are mi(ed and the complementar$ stic#$ ends for each plasmid are sealed b$ *NA Ligase% The p-L/ plasmid 0 which contains the genes for -3P! or -reen 3luorescent Protein! and the en2$me 45lactamase! which provides resistance to the antibiotic ampicillin 0 will be incorporated into the genome of the 6% Coli bacteria used in the lab% p-L/ is originall$ &from the bioluminescent jell$fish! Ae"uorea 7ictoria which allows the jell$fish to fluoresce and glow in the dar#% 6% coli can be transformed to+ ma#e the -3P protein and &e(press this gene+ which will &cause the 6% 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 demonstrate the effect of recombinant plasmids and transformation% B$ appl$ing ampicillin to a Petri dish with and without p-L/! we can see the success of an antibiotic against bacteria as well as the success of recombinant bacteria against an antibiotic% 9ince p-L/ harbors 45lactamase 0 a restriction en2$me which cuts the *NA of an antibiotic to render it ineffective 0 along with -3P! an$ bacteria with the p-L/ recombinant plasmid can better resist the antibiotic ampicillin% In our e(periment! the control treatment is the dish with bacteria! but lac#ing p-L/! ampicillin and agarose% /n this dish! we can see how the bacteria grow unimpeded or aided b$ an$ other substance% The e(perimental groups are the plate with bacteria! ampicilline and p-L/! the plate with onl$ bacteria and ampicillin! and the plate with ampicillin! agarose and p-L/% :$pothesis: ;hat plates will have growth and wh$<: The plates with p-L/! because the$ are resistant to antibiotics% ;hat plates will not have growth and wh$<: The plate with ampicillin and no p-L/! because the bacteria are compromised b$ an antibiotic and do not have a

recombinant plasmid% ;hat plates will glow under a 87 light and wh$<: plasmid contains the gene for glowing% 1esults '*ata = Anal$sis)

Plates with p-L/ because the

*iscussion>Conclusion To conduct this lab! we first too# two solutions of transformation solution and placed a starter colon$ of bacteria in each one% Then! to one solution! we added a p-L/ plasmid *NA solution% ;e then incubated both solutions! treated them with a heat shoc#! added LB nutrient broth to both and incubated them once more% Then! on two Petri dishes we spread the solution with p-L/! while on two more we spread the solution lac#ing p-L/% /n one p-L/ Petri dish we spread ampicillin on the other! we spread ampicillin and agarose% /n one Petri dish without p-L/ we spread ampicillin on the other! we spread nothing% 3inall$! we incubated the four Petri dishes four appro(imatel$ ?@ hours and let the bacteria grow% After the final incubation period! the dish with onl$ the initial solution and no p-L/ developed into a lawn of bacteria! coverin about ABC of the dish% The plate with ampicillin! but no p-L/! produced no bacteria% The dish with both p-L/ and ampcilin produced about B@A colonies of bacteria% The dish with ampicillin! agarose and p-L/ produced about ADE colonies of bacteria% The lac# of growth on the plate with onl$ ampicillin and the bacterial solution is due to the fact that ampicillin is an antibiotic which inhibits production of bacterial cell walls% Thus! no bacteria can be produced unless recombination occurs% The plate with onl$ bacteria and no p-L/ produced an entire lawn because the bacteria were able to grow unchec#ed b$ an antibiotic% The plate with p-L/ and ampicillin produced bacteria regardless of the antibiotics presence because the bacteria possess a plasmid that has undergone recombination and

therefore become resistant to the antibiotic% The plate with p-L/! agarose and ampicillin grew nearl$ one5and5a5half times the number of colonies as the plate with onl$ p-L/ and ampicillin because the sugar agarose is a nutrient for the bacteria which helps them divide at a "uic#er rate% The onl$ plate to glow was the plate with ampicillin! agarose and p-L/ a possible e(planation for wh$ this plate glowed when e(posed to 87 light but the plate with onl$ p-L/ and ampicillin did not is that the presence of agarose causes the operon that is responsible for glowing 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 the presence of an antibiotic% The transformation efficienc$ of BDF?%B? indicates that appro(imatel$ BDFG cells were transformed for ever$ one microgram of *NA% Possible sources of error in our e(periment include inaccuratel$ measuring the number of bacterial colonies that grew on each plate! insufficientl$ mi(ing the solutions of *NA after adding restriction en2$mes or not returning the mi(tures to ice "uic#l$ enough after during the heat shoc#% Literature Cited H*efinition of -enetic Transformation%H ,edicineNet% EI June DKKK% D? Apr% ?EEA Lhttp:>>www%medterms%com>script>main>art%asp<article#e$MKAEGN% HThe ,$ster$ Behind P-lo and -3P%H Chemistr$ and Biochemistr$ At ,C% ?EE?% The ,onmouth College Biochemistr$ 9taff% D? Apr% ?EEA Lhttp:>>department%monm%edu>chemistr$>chemistr$GGE>fall?EE?>nauclair>N%