Bacterial Transformation Lab Report Essay

BackroundThe plasmid pGLO contains an antibiotic-resistance gene, ampR, and the GFP gene is regulated by the control region of the genus Ara operon. ampicillin is an antibiotic that kills E. coli, so if E. coli, so if E. coli boothular telephones contain the ampicillin-resistance gene, the cells can survive exposure to ampicillin since the ampicillin-resistance gene encodes an enzyme that inactivates the antibiotic. Thus, alter E. coli cells containing ampicillin-resistance plasmids can easily be selected simply growing the bacteria in the presence of ampicillin-only the transformed cells survive. The ara control region regulates GFP expression by the addition of arabinose, so the GFP gene can be saturnine on and off by including or omitting arabinose from the culture medium.PurposeThe purpose of this testing ground was to understand bacterial shift key, how it occurs, and to make DNA glow.HypothesisIf the transformed E. coli is mixed with the ampicillin resistance gene, it wil l be fitting to grow in the ampicillin plates, but the non-transformed E.coli will not.MaterialsTwo microcentrifuge tubes500 uL of ice cold 0.05 CaCl2E. coli bacteriaA stereotypic malleable cringleA sterile P-20 micropipette10 uL of pAMP solutionA timerIceA water bath500 uL of Luria brothA spreading rod intravenous feeding platesIncubatorProcedureDay before lab1. Streak E. coli host cells for isolation.2. Prepare six seed plates.Day of lab1. Get two microcentrifuge tubes, which should for each one contain 200 uL of cold CaCl2 solution. Label maven tube with your initials and a (+) and the other tube with your initials and a (-).2. Transfer 2-4 large colonies using a sterile plastic curl up to each microcentrifuge tube and completely resuspend. Do not transfer any nutrient agar. perpetrate the tip of the loop into the CaCl2 solution and spin until there is not any cells on the loop.3. Close each of the tubes and put them in ice.4. Ask your teacher to use a P-20 micropipett e to add pGLO DNA to your fault mix.5. confer pGLO DNA to the (+) labeled microcentrifuge tube.6. Incubate some(prenominal) microcentrifuge tubes on ice for fifteen minutes.7. Take both tubes break of ice and immediately place in incubator at 42C for 90 seconds.8. by and by place both tubes back in the ice for two minutes.9. Add 200uL Luria Recovery Broth to both microcentrifuge tubes.10. Let both the tubes rest at room temperature for 10 minutes.11. During the 10 minutes, get the LB agar and LB+AMP agar plates ready. Mark your plates with the transformation tube mixture to use (+ or -), the lab group names, and the date on the top of the dishes.12. Add 100ul of the pGLO transformation cell mixture to the center of the agar surface of the corresponding LB agar and LB+AMP plates.13. Use a sterile plastic loop to distribute the cell suspension evenly on the plate by skating the loop back and forth across the LB agar plate several times.14. Use the same loop and technique to sprea d the same cell suspension (+) on the LB+AMP agar plates. lean ofthe sterile loop in a beaker of germicide.15. Repeat the procedure by spreading the (-) transformation cell mixture to each of the (-) labeled LB and LB+AMP plates. Be sure to use a fresh plastic loop for the None transformation mix.16. Stack your groups set of plates on top of one another and tape them together. The plates should be left upright position to allow the cell suspension to be absorbed by the agar.17. Place the plates in an inverted position (agar side on top) in a 37C bacterial incubation oven for overnight incubation (15-20 hrs.).Day later on lab1. Lower the clear-cuting in the room and use a long wave U.V. light to visualize the transformed cells that will glow due to the expression of the green or blue fluorescent proteins.DataLB+(Positive Control)LB-(Positive Control)LB/AMP+(Experimental)LB/AMP-(Experimental)Bacterial Growthlawnlawn3 coloniesNo increaseConclusionsThe bacteria treated with the pAM P solution developed a resistance to ampicillin and were able to grow on the ampicillin plate. Those that were not treated with the pAMP were not able to grow on this medium. The plates with no ampicillin served as a control to show how the bacteria would lookin normal conditions. Transformation is never in full effective, Only cells that are competent enough are able to take up the foreign DNA. Therefore, the ampicillin+ plates showed less growth that the control plate.Questions1. Record your observations about the color and growth (number of colonies) of bacteria on the Petri plates. If you ask so much bacterial growth that you cant count individual colonies, this is referred to as lawn.LB+(Positive Control)LB-(Positive Control)LB/AMP+(Experimental)LB/AMP-(Experimental)Bacterial Growthlawnlawn3 coloniesno growth2. Calculate the transformation efficiency of your transformation experiments. Transformation efficiency refers to the number of cells transformed per microgram (ug) of D NA. The transformation efficiency of my transformation experiments is 0.0125 cells transformed per microgram (ug) of DNA.