pGLO Transformation Lab

Purpose:

The purpose of this lab is to perform a genetic transformation of a gene for bioluminescence and to determine the degree of success in your efforts.


Pre-Lab Discussion:


Consideration 1: Can I genetically transform an organism? Which Organism?


1. To genetically transform an entire organism, you must insert the new gene into every cell in the organism. Which organism is more feasible for complete genetic transformation- a single-celled organism, or a multi-cellular organism?





2. Scientists often want to know if the genetically transformed organism can pass its new traits to its offspring and future generations. In order to determine this, which would be better to investigate -- an organism that develops and reproduces quickly, or an organism which develops and reproduces slowly?






3. Safety is another important consideration in selecting an organism for an experiment. What traits should the organism have or not have to be sure it will not harm the environment?










4. Based on the above considerations, which is the best choice for a genetic transformation experiment: a bacterium, earthworm, fish, or mouse? Explain your reasoning.













Consideration 2: How can I tell if cells have been genetically transformed?


1. The goal of genetic transformation is to change an organism’s phenotype. Before any change in the phenotype of an organism can be determined, the natural phenotype of the organism must be carefully studied. Look at the colonies of E. coli on your starter plates.


Record your observations in the following data table:

Trait

Obsevations

Number of colonies


size of the largest colony


size of the smallest colony


size of the majority of colonies


color of the colonies in normal lighting


distribution of the colonies on the plate

(draw a diagram)


visible appearance when viewed with UV light



Consideration 3: The genes


Bacteria often contain one or more plasmids, small circular pieces of DNA, in addition to the one large chromosome. Plasmid DNA usually contains genes for multiple traits. By using genetic engineering, genes that code for new traits can be inserted into a plasmid. In this lab, the pGLO plasmid carries the GFP gene that codes for the Green Fluorescent Protein. The genetically engineered plasmid can be used to transform bacteria to give them this new trait.


Materials:


E.coli starter plate Pipets Marking pen

2 poured agar plates Inoculation loops LB nutrient broth

Transformation solution(CaCl2) Foam micro tube holder Foam cup of crushed ice

Rehydrated pGLO plasmid 42°C water bath and thermometer 37°C incubator


Procedure:

  1. Label one closed micro test tube +pGLO and another –pGLO. Label both tubes with your names and place them in the foam tube rack.

  2. Open the tubes, and using a sterile transfer pipet, transfer 250 µL of transformation solution (CaCl2) into each tube.

  1. Place the tubes with the tube rack in the ice.

  2. Use a sterile loop to pick up a single colony of bacteria from your starter plate. Pick up the +pGLO tube and immerse the loop into the transformation solution at the bottom of the tube. Spin the loop between your index finger and thumb until the entire colony is dispersed in the transformation solution. Place the tube back in the tube rack in the ice. Using a new sterile loop, repeat for the –pGLO tube. Close the –pGLO tube.

  1. Examine the pGLO DNA solution with the UV lamp. Note your observations. Immerse a new sterile loop into the pGLO plasmid DNA stock tube and withdraw a loopful so that there is a film of the solution across the ring. Mix the loopful into the cell suspension of the +pGLO tube. Close the tube and return in to the rack on ice.

  1. Incubate the tubes on ice for 10 minutes. Make sure to push the tubes all the way down in the rack so that the tubes make contact with the ice.

  2. Label your two nutrient agar plates on the bottom: one +pGLO and the other –pGLO.

  3. Heat shock method: Using the foam rack as a holder, move both the +pGLO and –pGLO tubes into the water bath, set at 42°C, for exactly 50 seconds. Make sure that the tubes are all the way in the rack so that they touch the warm water. After 50 seconds, place both tubes back on ice. Incubate the tubes on ice for 2 minutes.

  4. Remove the rack containing the tubes from the ice and place them on the bench top. Open a tube and, using a new sterile pipet, add 250 µL of LB nutrient broth to the tube and recluse it. Repeat with a new sterile pipet for the other tube. Incubate the tubes for 10 minutes at room temperature.

  1. Tap the closed tubes with your finger to mix. Using a new sterile pipet for each tube, pipet 100 µL of the transformation and control suspensions onto the appropriate nutrient agar plates.

  1. Use a new sterile loop for each plate. Spread the suspension evenly around the surface of the LB nutrient agar by quickly skating the flat surface of a new sterile loop back and forth across the plate surface. DO NOT PRESS TOO DEEP INTO THE AGAR.

  1. Place the lids on the plates, stack them up and tape them together. Put your group name and class on the bottom of the stack. Place the stack of plates upside down in the 37°C incubator until the next day.




Review Questions:

Before collecting data and analyzing your results, answer the following questions.


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 prediction.






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





3. What is meant by a control plate? What purpose does a control serve?





Data Collection:


Observe the results you obtained from the transformation procedure under normal room lighting. Then, turn out the lights and hold the UV light over the plates. Record your observations in the data table below:

Observations of +pGLO plate

Observations of –pGLO plate

Draw a diagram of what you see on the plate



Amount of bacterial growth on the plate (relatively)



Color of bacteria



# of bacterial colonies (spots)



Under UV light





Analysis:

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








2. Recalling your observations when you shined the UV light onto a sample of original pGLO plasmid DNA and your results from the transformed plate,


a) which of the two possible sources of the fluorescence can now be eliminated?





b) what is the probable source of the fluorescence?





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
























Note: This lab was modified from the Bio-Rad pGLO Bacterial Transformation Kit manual.