Lesson 3 Review Questions

By: Jathuya Peranantharuban and Leeza Yackballick

What’s Glowing?

1.                   Recall what you observed when you shined the UV light source onto a sample of

original pGLO plasmid DNA and describe your observations.

When the UV light was shined on the sample of the original pGLO plasmid DNA,  no fluorescence colour was observed, and did not change in appearance under the UV light.

2.                   Which of the two possible sources of the fluorescence can now be eliminated?

The original bacteria and the pGLO plasmids DNA can be eliminated since no fluorescence colour was observed under the UV light.

3.                   What does this observation indicate about the source of the fluorescence?

This observation indicates that the source of of fluorescence is related to the protein in the DNA of the transformed plasmids. The fluorescence green light was only observed in the DNA of the bacteria that contains the pGLO plasmid DNA, LB nutrient, Ampicillin and Arabinose.  This observation indicates the source of fluorescence is from the gene that codes for green fluorescent protein from the bioluminescent jellyfish Aequorea victoria. This gene allows the transformed bacteria to glow a bright green colour under ultraviolet light.

4.                   Describe the evidence that indicates whether your attempt at performing a genetic transformation was successful or not successful.

The evidence that would indicate whether our attempt at performing a genetic transformation was successful or not depends on the end observations. If the transformed bacteria display the desired characteristic then we have successfully performed genetic transformation. In this case,  if the plate with +pGLO LB/amp/ara and +pGLO LB/amp have colonies of bacteria, and the colonies on the +pGLO LB/amp/ara plate should fluoresce bright green under UV light. If the experiment was unsuccessful then colonies of bacteria will not be present on the  +pGLO LB/amp/ara and +pGLO LB/amp plates.  Errors that may affect the results include not transferring appropriate number of colonies to the plates or mistakes in following the instructions as indicated in the procedure.

The Interaction between Genes and Environment

Some E.coli did grow on the LB plate that does not contain ampicillin  or arabinose.

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

From our results, we cannot tell if these bacteria are ampillicin restistent by looking at them on the LB plate.  We are not able to distinguish the colonies that are ampicillin resistant and those that are ampicillin sensitive since they look similar. The bacteria on the LB plate and the bacteria on the LB/amp plate look alike, so therefore there is not a physical feature to indicate whether they are ampicillin resistant.

2.                   How would you change the bacteria’s environment to best tell if they are ampicillin resistant?

To best tell if they are ampicillin resistant, we can change the bacteria’s environment. To test whether the bacteria are resistant to ampicillin, some bacteria  can be transferred from the LB plate to the LB/amp plate. If the bacteria are able to grow on the LB/amp plate, this would show the bacteria are resistant to ampicillin, whereas if the bacteria do not survive, they are ampicillin sensitive. The plasmids that are able to survive contain the gene resistant to the antibiotic, and therefore when the plasmids are placed in the new environment with the antibiotic, the colonies can continue to grow.

3.                   Very often an organism’s traits are caused by a combination of its genes and the environment it lives in. 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).

The two factors that must be present is the appropriate solutions and environment. The sugar arabinose must be present to activate the GFP gene.  This gene is next to the arabinose promoter, so it becomes active in the presence of arabinose. In addition, UV light is necessary to cause the GFP protein to fluoresce.

b. What do you think each of the two environmental factors you listed above are doing to cause the genetically transformed bacteria turn green?           

When these two factors are present, the transformed bacteria display the gene from the jellyfish.  The original plasmid contained three different genes ,ara A, ara B, and ara D that code for three enzymes needed to convert arabinose to a form that can be used. A fourth gene, araC, codes for a protein that acts to control the structural genes. When arabinose is present, it initiates transcription by promoting the binding of RNA polymerase. The arabinose interacts with araC and causes araC to change its shape. In the transformed plasmids, the promoter (P_BAD) and the araC gene are present but the ara A, ara B, and ara D genes are replaced by the GFP gene. Therefore, in the presence of arabinose, araC protein promotes the binding of RNA polymerase and GFP is produced. Furthermore, the long-wave UV light give the gene the energy to fluoresce the green colour.

c.  What advantage would there be for an organism to be able to turn on or off particular genes in response to certain conditions?

Gene regulation is an important feature that allows organisms to survive and prevent producing unnecessary proteins. This allows the organism to produce proteins only when needed and according the their environment. Moreover, control of the gene is important to allow a cell to create the gene products it needs when it needs them. This gives cells the flexibility to adjust to a new environment, external signals, damage to the cell, food, etc. Examples include the Lac operon in E.Coli, a negative control system, that breaks down lactose when present. Another example is a positive control system since E.coli need tryphospho to produce proteins, the Trp operon is usually “on”.