Transformation Lab Answers

1. On which of the plates would you expect to find bacteria most like the original non-transformed E. coli colonies you initially observed? Explain your predictions. - I’d expect to find the non-transformed bacteria on the pGLO+ LB/AMP/ARA dish, without the introduction of arabinose. I think this because the bacterias would still be intact due to it not being killed by the ampicillin, and therefore look still like the control plate, just without the glow. 2. If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be located? Explain your predictions. - They’d be on the +pGLO plate, because the cells would survive long enough to be transformed, because the transformation gives them the ability to survive the ampicillin. 3. Which plates should be compared to determine if any genetic transformation has occurred? Why? - I think the +pGLO plates should be compared against each other (one with arabinose, one without). I say this because then it is a fair comparison, because they both have the pGLO DNA. 4. What is meant by a control plate? What purpose does a control serve? - A control plate exists to show a further difference in the final product of the experiment, a meaning of comparison between different variables added. In this case, the difference between arabinose and no arabinose, or ampicillin versus no ampicillin. PAGE 43 1. 2. How much bacterial growth do you see on each plate, relatively speaking? - On the +pGLO plate without arabinose, there’s some bacterial growth, but less dense growth compared to the -pGLO plate with no ampcillin (presumably just e coli and the broth?). On the +pGLO plate with arabinose, there’s more glowing bacterial growth it looks like, but the spots are smaller. On the -pGLO plate with ampicillin added, there is no bacterial growth. 3. What color are the bacteria? - When looking at the +pGLO plate with arabinose added, the e coli is glowing green. The other bacterias shown on the plates are just a greyish color. 4. How many bacterial colonies are on each plate (count the spots you see). - On the -pGLO plate with no ampcillin, there is one very large spot and two small spots. On the -pGLO plate with ampicillin, there is nothing. On the +pGLO plate without arabinose, there’s about 50 spots. On the +pGLO plate with arabinose, there’s like 100+ spots. 1. Which of the traits that you originally observed for E. coli did not seem to become altered? In the space below list these untransformed traits and how you arrived at this analysis for each trait listed. - One unaltered trait is the shape of the grown e coli that was untouched on the broth plate (-pGLO). You can assume although all of the e coli on the next plate died due to the ampicillin, the shape did not change. 2. Of the E. coli traits you originally noted, which seem now to be significantly different after performing the transformation procedure? List those traits below and describe the changes that you observed. - One trait that is very different from the control “blob” on the -pGLO plate with broth is the +pGLO plate with broth and ampicillin. There are colonies now, not a blob, because the majority of the “blob” died when the ampicillin was added, and the colonies that are left represent the e coli's who were able to transform and gain immunity to the ampicillin. The untransformed -pGLO plate with ampicillin shows that they all died. 3. If the genetically transformed cells have acquired the ability to live in the presence of the antibiotic ampicillin, then what might be inferred about the other genes on the plasmid that you used in your transformation procedure? - The other genes on the transformed plasmid have presumably also acquired the ability to live amongst the ampicillin. 4. From the results that you obtained, how could you prove that the changes that occurred were due to the procedure that you performed? - Because e coli doesn’t naturally glow on its own, and did not glow without the presence of arabinose. 1. Recall what you observed when you shined the UV light onto a sample of original pGLO plasmid DNA and describe your observations. - Because the DNA has the pGLO gene in it, the UV light would make it glow green, but ONLY if arabinose is there too. 2. Which of the two possible sources of the fluorescence can now be eliminated? - The ampicillin, which was in the original +pGLO plate, did not make the sample glow green. However, the ampicillin and the +pGLO along with arabinose in the plate did make it glow. So ampicillin can be eliminated as a glow agent. 3. What does this observation indicate about the source of the fluorescence? - +pGLO plates will not glow without arabinose. 4. Describe the evidence that indicates whether your attempt at performing a genetic transformation was successful or not successful. - The evidence that this transformation was successful is that the +pGLO sample did glow when the rest of the environment was correct, and that the e coli sample died when it didn’t have the beta-lactamase (in the pGLO DNA) to help it survive in the ampicillin, but the sample that did have the beta-lactamase in it survived in the ampicillin. That is proof that the experiment was successful. 1. From your results, can you tell if these bacteria are ampicillin resistant by looking at them on the LB plate? Explain your answer. - Yes, because the bacteria that are ampicillin resistant are there, and therefore visible. 2. How would you change the bacteria’s environment—the plate they are growing on—to best tell if they are ampicillin resistant? - You could put ampicillin on the plate and see if the bacteria die or not. If they do not die, they’re obviously resistant. 3. Very often an organism’s traits are caused by a combination of its genes and its environment. Think about the green color you saw in the genetically transformed bacteria: a. What two factors must be present in the bacteria’s environment for you to see the green color? (Hint: one factor is in the plate and the other factor is in how you look at the bacteria). - One factor is the arabinose sugar that must be there to help the GFP gene be expressed. The other factor is UV light. b. What do you think each of the two environmental factors you listed above are doing to cause the genetically transformed bacteria to turn green? - The UV light gets absorbed by the GFP protein by some amino acids inside of it, then emits the light back out as the green light that we see. The arabinose sugar just makes the regulatory system inside the gene make RNA, which in turn makes more GFP, and the more there is, the more green it will look. c. What advantage would there be for an organism to be able to turn on or off particular genes in response to certain conditions? - For example, imagine an animal with a lot of GFP in it that made it always glow green. That animal would be an insanely easy target for predators and would have zero camouflage abilities. If the animal had the ability to turn off that gene at least at night, then it would have a way better chance of survival. Or if for some reason having the GFP gene expressed (glowing) caused cells to heat up and therefore produce heat, an animal living in cold conditions having the ability to turn on that gene that made it warmer could be beneficial towards surviving harsh winters.