Bacterial Transformation Lab: pGLO

What is the total volume of reagent in mL?

0.250mL or 250 microliters

What is a bacterial colony?

A bacterial colony is a large group or cluster of bacterial cells that originated from a single, clonal cell.

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

A bacterium would be the best host organism. Bacteria are small, single-celled organisms which reproduce quickly and easily.

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

A control plate is a guide that is used to help you interpret the experimental results. In this experiment, both (-) pGLO plates are control plates. The LB/amp control plate can be compared to the LB/amp (+)pGLO plate. This comparison shows that genetic transformation produces bacterial colonies that can grow on ampicillin (due to the uptake of the pGLO plasmid and the expression of the ampicillin resistance gene). The (-) pGLO/LB control plate can be compared to any of the LB/amp plates to show that plasmid uptake is required for the growth in the presence of ampicillin. The (-) pGLO LB/amp plate shows that the starter culture does not grow on the LB/amp plate. Without this control, one would not know if the colonies on the LB/amp (+) pGLO plate were really transformants.

To genetically transform an entire organism, you must insert the new gene(s) 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?

A single-celled organism would be the best recipient for a genetic transformation because it contains only one cell which needs to take up the new gene.

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

A successful experiment will be represented by the presence of colonies on the (+) pGLO LB/amp and (+) pGLO LB/amp/ara plates and the absence of colonies on the (-) pGLO LB/amp plate. Moreover, the colonies on the LB/amp/ara plate should fluoresce green. An unsuccessful experiment will show an absence of colonies on the (+) pGLO LB/amp and (+) pGLO LB/amp/ara plates. This could be a result of not adding a loopful of plasmid to the (+) pGLO tube or not adding a colony of bacteria to the (+) pGLO tube

Scientists often want to know if the genetically transformed organism can pass its new traits on to 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?

An organism which reproduces quickly. Fast production of offspring or new progeny will allow you to quickly assess if the new trait has been passed on.

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

Antibiotics usually kill bacteria (are bacteriocidic) or inhibit their growth (bacteriostatic). Thus, there should be few, if any, bacterial colonies present on the ampicillin plate. The presence of any colonies on the ampicillin plate would suggest that those bacteria are resistant to the antibiotic ampicillin.

Which of the traits that you originally observed for E. coli did not seem to become altered? In the space below, list these non-transformed traits and how you arrived at this analysis for each trait listed.

Bacteria are a whitish color. Colony size is similar both before and after transformation.

On which of the plates would you expect to find bacteria most like the original untransformed E. coli colonies you initially observed? Explain your prediction.

Bacteria which resemble the non-transformed will be found on the LB/(-) pGLO plate. These bacteria were removed from the starter plate, did not have any plasmid added to them, and were replated on an LB plate. Thus, they are virtually identical to the non-transformed starter colonies.

Genetic transformation occurs when a cell takes up and expresses a new piece or foreign ____________, often a circular plasmid.

DNA

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

Equal amounts of cells could be plated on two different LB nutrient agar plates, one which contains just LB nutrient agar and one which contains LB nutrient agar ampicillin. The growth of E. coli on the two plates could be compared. If ampicillin negatively affects the growth of E. coli, then there should be fewer colonies of bacteria on that plate. If ampicillin has no effect, there should be approximately equal numbers of colonies 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 different conditions and prevents wasteful overproduction of unneeded proteins. Good examples of highly regulatable genes are the enzymes which break down carbohydrate food sources. If the sugar arabinose is present in the growth medium it is beneficial for bacteria to produce the enzymes necessary to catabolize the sugar source. Conversely, if arabinose is not present in the nutrient media, it would be very energetically wasteful to produce the enzymes to break down arabinose.

Can you predict what would happen if you took one of the green colonies from the LB/amp/ara plate and streaked it onto an LB/amp plate? Conversely, what would happen if you took a white colony from the LB/amp plate and streaked it onto an LB/amp/ara plate? Explain your answer.

If a green colony under UV light was streaked onto an LB/amp plate, the resulting colonies would be white with no fluorescence. This plate does not contain arabinose which is needed to induce expression of the GFP gene and generate green fluorescent colonies. If a white colony was streaked onto an LB/amp/ara plate, the resulting colonies would be green. This plate contains arabinose which induces expression of the GFP gene and generates green fluorescent colonies.

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

No. You cannot tell if the bacteria are ampicillin resistant just by looking at them. Both types of bacteria (those that are ampicillin resistant and those that are ampicillin sensitive) look similar when cultured—think about the colonies on the LB starter plate and the colonies on the +pGLO LB/amp plate.

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

The LB/amp (-) pGLO and the LB/amp (+) pGLO plates should be directly compared. Cells which were not treated with DNA (-pGLO) should not be expressing the ampicillin resistance gene and will not grow on the LB/amp plates. Cells which were treated with DNA (+pGLO) should contain the pGLO plasmid and should express the ampicillin resistance gene—the corresponding LB/amp plate will contain transformed bacterial colonies.

What was the purpose of rupturing or lysing the bacteria?

The bacteria need to be ruptured in order to release the GFP/plasmid DNA, which can then be purified using column chromatography.

What color are the bacteria?

The bacteria on the (+) pGLO LB/amp plate and the (-) pGLO LB plates should be whitish. The bacteria on the (+) pGLO LB/amp/ara plate should appear whitish when exposed to normal, room lighting, but fluoresce bright green upon exposure to the long-wave UV light.

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

The best test would be to take some of the bacteria growing on the LB plate and streak them on an LB/amp plate. If the bacteria are viable on the LB/amp plate, then they are resistant to ampicillin. If no bacterial colonies survive, then they were not ampicillin resistant (they were ampicillin sensitive).

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

The best way is to compare the control to the experimental plates. Cells that were not treated with the plasmid (LB/amp (-) pGLO and LB/amp/ara (-) pGLO plates) could not grow on ampicillin, whereas cells that were treated with the plasmid (LB/amp (+) pGLO and LB/amp/ara (+) pGLO plate) can grow on the LB/amp plate. Thus, the plasmid must confer resistance to ampicillin

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.

The colonies on the LB/amp/ara plate fluoresce green under UV light, and the transformed colonies can grow on ampicillin resistance

Recall that the goal of genetic transformation is to change an organism's traits (phenotype). Before any change in the phenotype of an organism can be detected, a thorough examination of its usual (pre-transformation) phenotype must be made. Look at the colonies of E. coli on your starter plates. List all observable traits or characteristics that can be described.

The color of colonies, number of colonies, distribution of colonies on the plate.

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

The organism should not produce any toxins or compounds which could make people sick. The organism should grow vigorously in the lab environment, but should not be able to survive outside the laboratory. The organism should not be able to infect plants or animals.

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

The pGLO plasmid DNA and the original bacteria can be eliminated from providing the fluorescent source.

If the genetically transformed cells have acquired the ability to live in the presence of the antibiotic ampicillin, then what can be inferred about the other genes on the plasmid that were involved in your transformation procedure?

The plasmid must express a gene for ampicillin resistance (the protein product of the bla gene codes for beta-lactamase, the protein that breaks down ampicillin).

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

The plasmid sample (LB/amp +pGLO) did not fluoresce.

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

The source of fluorescence is probably from some protein that the plasmid encodes from the addition of arabinose, namely GFP.

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

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

The sugar arabinose turns on expression of the GFP gene by binding to a regulatory protein, araC, which sits on the PBAD promoter. When arabinose is present, it binds to araC, consequently changing the conformation of araC which facilitates transcription of the gene by RNA polymerase (see detailed description in Appendix D). When exposed to UV light, the electrons in GFP's chromosphere are excited to a higher energy state. When they drop down to a lower energy state they emit a longer wavelength of visible fluorescent green light at 509 nm.

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

The transformed cells are found on the LB/amp and LB/amp/ara plates. Genetically transformed cells have taken up the pGLO plasmid which expresses the ampicillin resistance gene—these cells can survive on the plates which contain ampicillin.

Can you explain why the bacterial cells' outer cell wall ruptures when the cells are frozen? What happens to an unopened soft drink when it freezes?

When a bacterial cell freezes, the volume of cytoplasm expands. The expansion puts pressure on the weakened cell wall, which then ruptures from the pressure.

Look again at your four plates. Do you observe some E. coli growing on the LB plates which do not contain ampicillin/arabinose?

Yes. The bacteria that did not receive the plasmid are growing on a plain LB plate.

pGlo plasmids, when taken up by a bacteria, will code for

a green fluorescence protein GFP

What are 3 real-world links for the study/use of genetic GFP?

study of biological processes, observation of cell movement, use of GFP as a visual marker