Ap Biology Transformation Lab Report 2j4x2l

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 consequences that come of transformation; and to understand the importance of transformation in prokaryotic and eukaryotic life cycles. Background: Transformation is the “process by which the genetic material carried by an individual cell is altered by the incorporation of foreign (exogenous) DNA into its genome” (MedicineNet.com, “Definition of Genetic transformation”). Transformation in bacterial cells occurs when the cell incorporates naked DNA into its genetic material; in a laboratory setting, this is encouraged by placing the mixtures of transformation solution and plasmid DNA (in +pGLO tube only) on ice, then rapidly transferring them to a hot water bath for about fifty seconds, and then placing them back on ice again – this procedure is called heat shock and “increases the permeability of the cell membrane to DNA” (lab directions). The agent which the new genetic material is incorporated into is the bacterial plasmid. A plasmid is a circular deoxyribonucleic acid (DNA) molecule that replicates independently of the bacterial chromosome and often is the avenue for which a bacteria gains resistance to an antibiotic. Recombinant plasmids are those which have DNA from two or more sources incorporated into a single plasmid. To make recombinant plasmids, two different plasmids are cut with the same restriction enzyme: this restriction enzyme only cuts at particular restriction sites, so the type of cut it makes in one plasmid will be the same type of cut in another plasmid. The cut must produce “sticky ends” so that the plasmid DNA can bind to any other plasmid DNA with complementary base pairs. Once cut, the two plasmids are mixed and the complementary sticky ends for each plasmid are sealed by DNA Ligase. The pGLO plasmid – which contains the genes for GFP, or Green Fluorescent Protein, and the enzyme ß-lactamase, which provides resistance to the antibiotic ampicillin – will be incorporated into the genome of the E. Coli bacteria used in the lab. pGLO is originally “from the bioluminescent jellyfish, Aequorea Victoria which allows the jellyfish to fluoresce and glow in the dark. E. coli can be transformed to” make the GFP protein and “express this gene” which will “cause the E. coli to glow green when exposed to UV light” (The Mystery Behind pGlo and GFP). The aforementioned antibiotic ampicillin is used in this lab to demonstrate the effect of recombinant plasmids and transformation. By applying ampicillin to a Petri dish with and without pGLO, we can see the success of an antibiotic against bacteria as well as the success of recombinant bacteria against an antibiotic. Since pGLO harbors ß-lactamase – a restriction enzyme which cuts the DNA of an antibiotic to render it ineffective – along with GFP, any bacteria with the pGLO recombinant plasmid can better resist the antibiotic ampicillin. In our experiment, the control treatment is the dish with bacteria, but lacking pGLO, ampicillin and agarose. On this dish, we can see how the bacteria grow unimpeded or aided by any other substance. The experimental groups are the plate with bacteria, ampicilline and pGLO, the plate with only bacteria and ampicillin, and the plate with ampicillin, agarose and pGLO. Hypothesis: What plates will have growth and why?: The plates with pGLO, because they are resistant to antibiotics. What plates will not have growth and why?: The plate with ampicillin and no pGLO, because the bacteria are compromised by an antibiotic and do not have a

recombinant plasmid. What plates will glow under a UV light and why?: plasmid contains the gene for glowing.

Plates with pGLO because the

Results (Data & Analysis)

Discussion/Conclusion To conduct this lab, we first took two solutions of transformation solution and placed a starter colony of bacteria in each one. Then, to one solution, we added a pGLO plasmid DNA solution. We then incubated both solutions, treated them with a heat shock, added LB nutrient broth to both and incubated them once more. Then, on two Petri dishes we spread the solution with pGLO, while on two more we spread the solution lacking pGLO. On one pGLO Petri dish we spread ampicillin; on the other, we spread ampicillin and agarose. On one Petri dish without pGLO we spread ampicillin; on the other, we spread nothing. Finally, we incubated the four Petri dishes four approximately 24 hours and let the bacteria grow. After the final incubation period, the dish with only the initial solution and no pGLO developed into a lawn of bacteria, coverin about 85% of the dish. The plate with ampicillin, but no pGLO, produced no bacteria. The dish with both pGLO and ampcilin produced about 548 colonies of bacteria. The dish with ampicillin, agarose and pGLO produced about 810 colonies of bacteria. The lack of growth on the plate with only 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 only bacteria and no pGLO produced an entire lawn because the bacteria were able to grow unchecked by an antibiotic. The plate with pGLO 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 pGLO, agarose and ampicillin grew nearly one-and-a-half times the number of colonies as the plate with only pGLO and ampicillin because the sugar agarose is a nutrient for the bacteria which helps them divide at a quicker rate. The only plate to glow was the plate with ampicillin, agarose and pGLO; a possible explanation for why this plate glowed when exposed to UV light but the plate with only pGLO and ampicillin did not is that the presence of agarose causes the operon that is responsible for glowing to become activated in order to break down agarose and make it usable by the bacterial cell. We know transformation occurred because there would be no bacterial colonies on the plate with both pGLO 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 efficiency of 5162.52 indicates that approximately 5163 cells were transformed for every one microgram of DNA. Possible sources of error in our experiment include inaccurately measuring the number of bacterial colonies that grew on each plate, insufficiently mixing the solutions of DNA after adding restriction enzymes or not returning the mixtures to ice quickly enough after during the heat shock. Literature Cited "Definition of Genetic Transformation." MedicineNet. 07 June 1999. 12 Apr. 2008 . "The Mystery Behind PGlo and GFP." Chemistry and Biochemistry At MC. 2002. The Monmouth College Biochemistry Staff. 12 Apr. 2008 .