Lesson 3: Review Questions

What's Glowing?

1. Recall what you observed when you shined the UV light onto a sample of original pGLO plasmid DNA and describe your observation.  (Christina)
   
   
   When we shined the UV light onto the negative pGLO plasmid DNA we did not see anything significant happen and the colonies were unaffected. When we shined the UV light on the positive pGLO plasmid DNA we saw the colonies in the plates fluoresce a green colour. This shows that protein is present in the plate and it fluorescing due to it.
   
   
2. Which of the two possible sources of the fluorescence can now be eliminated? (Anisha)
   
   After observing our results, there are two possible sources of the fluorescence which can now be eliminated. These sources include the pGLO plasmid DNA and the original bacterial cells. 
   
   
3. What does this observation indicate about the source of the fluorescence? (Athira)
   
   
   The source of fluorescence is from the plasmid. The pGLO plasmid has a green fluorescent protein that allows the transformed E.coli to glow under UV light.
   
   
4. Describe the evidence that indicates whether your attempt at performing a genetic transformation was successful or not successful.  (Samar)
   
   
   

   A somewhat successful experiment was represented by the presence of colonies within the (+) LB/amp/ara plate. The (+) LB/amp/ara plate also fluoresced green under the presence of UV light. In turn, the (-) LB/amp had no sign of colonies, which further proved the success of the experiment. One factor of the experiment that did not help the success of the experiment was the presence of colonies among the (-) LB/Amp plate and the glowing of the colonies under the UV light. In addition, the (+) LB/Amp plate showed no sign of colonies. There may have been an error within the transfer of loops that may have caused the success rate of this experiment to lower.

The Interaction between Genes and Environment

1. From your results, can you tell if these bacteria are ampicillin resistant by looking at them on the LB plate? Explain your answer.  (Anisha)
   
   

   It is not possible to tell if the bacteria are ampicillin resistant by looking at them on the LB plate. This is because both types of bacteria, those which are ampicillin resistant and those that are not, look very similar to each other. This is visible when comparing the appearance of the bacteria on the –pGLO LB plate which do not have the gene for ampicillin resistance and the bacteria on the +pGLO LB/amp plate. Both types of bacteria look very similar to each other, and therefore, cannot easily be distinguished as to which one contains the gene for ampicillin resistance.
   
   
2. How would you change the bacteria’s environment-the plate they are growing on-to best tell if they are ampicillin resistant?  (Athira)

   
   

   Fill it with ampicillin to see if they will be killed by the antibiotic.
   
   
3. 
   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).  (Samar)
   
   
   One factor within the plate that must be present in the bacteria’s environment for one to observe the expression of the GFP gene is the sugar arabinose in the agarose plate. Another factor that must be present in the bacteria’s environment includes the UV light, allowing the GFP protein within the bacteria to emit a fluorescent glow.
   
   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?  (Arrjun)
   
   
   The sugar arabinose turns on the expression of the green fluorescent protein by binding to a regulatory protein that is sitting on the promoter region. When arabinose is present it binds to regulatory protein, therefore changing its shape, facilitating the transcription of the gene by RNA Polymerase.
   
   c. What advantage would there be for an organism to be able to turn on or off particular genes in response to certain conditions?  (Christina)
   
   
   The advantage for an organism to be able to turn on or off particular genes in response to certain conditions would allow the gene to adapt in different conditions and would be able to prevent overproducing of unnecessary proteins.