pGLO Transformation

Genetic Transformation

"Change caused by genes" Involves the insertion of a gene into an organism in order to change the organism's trait Goal is to change an organism's phenotype

Transformation Efficiency

=Total number of colonies growing on the agar plate/amount of DNA spread on the agar plate A quantitative measurement used to help determine how well the transformation is working. Represents how effective you were in getting DNA molecules into bacterial cells

Based on the above considerations, which would be the best choice for a genetic transformation: a bacterium, earthworm, fish, or mouse?

A bacterium would be the best choice for a genetic transformation. They are well suited because they consist of a single cell and they reproduce and develop quickly.

Plasmid

A circular piece of DNA that usually contains genes for one or more traits that may be beneficial to bacterial survival.

What is meant by a control plate? What purpose does the control serve?

A control plate is a guide used to help interpret the experimental results. Both the -pGLO plates serve as controls to be compared to the +pGLO plates

Gene

A piece of DNA which codes for a protein that gives an organism a particular trait

What would you expect your experimental results to indicate about the effect of ampicillin on the E. coli cells?

Antibiotics kill bacteria or inhibit their growth. There should be very little, to no growth on the ampicillin pate. If any bacteria grows on the ampicillin plate, it would suggest that the bacteria is resistant to the antibiotic.

What do you think each of the 2 environmental factors are doing to cause the bacteria to turn green?

Arabinose turns on the expression of the GFP gene by binding to the regulatory protein, araC. When present, arabinose binds to araC and changes its conformation to facilitate transcription of the gene by RNA polymerase. Exposure to UV light causes GFP to resonate and give off energy in the form of green light.

What color are the bacteria.

Bacteria are white on all plates where it is present. Bacteria on the LB/amp/ara plate glows green under UV light

What happens after the transformation procedure?

Bacteria express their newly acquired gene and produce the fluorescent protein, which causes them to glow green under UV light.

How is the gene for GFP switched on in transformed cells?

By adding the sugar arabinose to the cells' nutrient medium.

Which of the traits that you originally observed for E. coli did not seem to become altered?

Color -- bacteria are whitish Colony size -- colony size is similar before and after transformation

OF the E. coli traits you originally noted, which seem now to be significantly different after performing the transformation procedure?

Color -- colonies on the LB/amp/ara plate fluoresce green under UV light Ampicillin -- transformed colonies can grow on ampicillin resistance

pGLO plasmid

Encodes the gene for GFP and a gene for resistance to the antibiotic ampicillin

Describe how you could use two LB/agar plates, some E. coli and some ampicillin to determine how E. coli cells are affected by ampicillin

Equal amounts of E. coli could be plated on two different LB/agar plates. One would only have the LB nutrient and one would contain the LB nutrient and ampicillin. Then you would compare E. coli growth on both plates

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 allows for adaptation to differing conditions and prevents the overproduction of unnecessary proteins.

Selection for cells that have been transformed with pGLO DNA

Is accomplished by growth on ampicillin plates

How much bacterial growth do you see on each plate, relatively speaking?

No growth on the LB/amp -pGLO. Lawn of bacteria on LB -pGLO plate. Multiple colonies on LB/amp and LB/amp/ara +pGLO plates

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

No, you can't just tell by looking at them. Bacteria that are resistant and those that aren't look similar.

Observations that can be used to determine if any genetic transformation has occurred

Number of colonies Size of the largest/smallest/majority of colony/ies Color of the colonies Distribution of the colonies on the plate Visible appearance when viewed with UV light The ability of the cells to live and reproduce in the presence of an antibiotic such as ampicillin

Scientists often want to know if the genetically transformed organism can pass its new traits onto its offspring and future generations. To get this information which would be a better candidate for your investigation, an organism in which each new generation develops and reproduces quickly, or one which does this more slowly?

One that develops and reproduces quickly, because results can be recorded immediately. It saves time and we can more quickly see offspring.

On which of the plates would you expect to find bacteria most like the original non-transformed E. coli colonies you initially observed?

The LB -pGLO plate, because the bacteria here did not receive any plasmid. They are identical to the starter E. coli.

Which plates should be compared to determine if any genetic transformation has occurred? Why?

The LB/amp -pGLO and the LB/amp +pGLO should be compared. Cells that did not get treated with DNA should not express resistance and shouldn't grow on the plate. Cells treated with the DNA should contain the pGLO plasmid and should express ampicillin resistance. This plate will contain bacterial colonies.

Real Life Source of Green Fluorescent Protein (GFP)

The bioluminescent jellyfish, Aequorea victoria

To genetically transform an entire organism, you must insert the new gene into every cell in the organism. Which organism is better suited for total genetic transformation -- one composed of many cells, or one composed of a single cell?

The organism better suited for total genetic transformation is one composed of a single cell, because you would only need to insert the gene into one cell rather than many.

Safety is another important consideration in choosing an experimental organism. What traits or characteristics should the organism have/not have to be sure it will not harm you or the environment?

The organism shouldn't produce any toxins or compounds that can infect humans. The organism should have the ability to grow at an optimal rate in the lab, but not in conditions outside of the lab environment.

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

The pGLO plasmid DNA and the original bacteria can now be eliminated

Recall what you observed when you shined the UV light onto a sample of original pGLO plasmid DNA and describe your observations.

The plasmid DNA did not fluoresce

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 plasmid must express a gene for ampicillin resistance

What does the observation indicate about the source of fluorescence?

The source of the fluorescence is from a protein that the plasmid encodes.

What 2 factors must be present in the bacteria's environment for you to see the green color?

The sugar arabinose in the agarose plate is needed to turn on the expression of the GFP gene. UV light is needed to cause the GFP protein in the bacteria to fluoresce.

If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be located?

The transformed cells would be found on the LB/amp +pGLO and the LB/amp/ara +pGLO plates. The genetically transformed bacterial cells took up the plasmid that expresses the ampicillin resistance gene, so they can survive on the plates with ampicillin

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

There was a presence of colonies on the +pGLO plates. No colonies grew on the -pGLO LB/amp plate. Colonies on the LB/amp/ara plate fluoresce green

Act of Transformation

To move the pGLO plasmid .DNA through he cell membrane you will: 1. Use a transformation solution containing calcium chloride. 2. Carry out a procedure referred to as heat shock For transformed cells to grow in the presence of ampicillin you must: 3. Provide them with nutrients and a short incubation period to begin expressing their newly acquired genes.

Purpose of pGLO Lab

To perform a genetic transformation to transform bacteria with a gene that codes for Green Fluorescent Protein, and determine how successful our genetic alteration of the organism was.

What do transformed cells appear like?

White on plates not containing arabinose. Fluorescent green under UV light when there is arabinose.

Look again at your four plates. Do you observe some E. coli on the LB plate that does not contain ampicillin or arabinose.

Yes, the bacteria on the plain LB plate that did not receive the plasmid

From the results that you obtained, how could you prove that the changes that occurred were due to the procedure that you performed?

You can prove this by comparing the control and experimental plates. Cells not treated with the plasmid couldn't grow on ampicillin, but the cells that were treated with the plasmid could grow.

Hw would you change the bacteria's environment -- the plate they are growing on -- to best tell if they are ampicillin resistant?

You could take bacteria growing on the LB plate and streak them onto the LB/amp plate. If they are viable, they are resistant. If no colonies survive, they were not resistant.