Student Manual pGLO Transformation Lesson 1 Introduction to Transformation In this lab you will perform a procedure known as genetic transformation. Remember that a gene is a piece of DNA which provides the instructions for making (codes for) a protein. This protein gives an organism a particular trait. Genetic transformation literally means “change caused by genes,” and involves the insertion of a gene into an organism in order to change the organism’s trait. Genetic transformation is used in many areas of biotechnology. In agriculture, genes coding for traits such as frost, pest, or spoilage resistance can be genetically transformed into plants. In bioremediation, bacteria can be genetically transformed with genes enabling them to digest oil spills. In medicine, diseases caused by defective genes are beginning to be treated by gene therapy; that is, by genetically transforming a sick person’s cells with healthy copies of the defective gene that causes the disease. You will use a procedure to transform bacteria with a gene that codes for Green Fluorescent Protein (GFP). The real-life source of this gene is the bioluminescent jellyfish Aequorea victoria . Green Fluorescent Protein causes the jellyfish to fluoresce and glow in the dark. Following the transformation procedure, the bacteria express their newly acquired jellyfish gene and produce the fluorescent protein, which causes them to glow a brilliant green color under ultraviolet light. In this activity, you will learn about the process of moving genes from one organism to another with the aid of a plasmid. In addition to one large chromosome, bacteria naturally contain one or more small circular pieces of DNA called plasmids. Plasmid DNA usually contains genes for one or more traits that may be beneficial to bacterial survival. In nature, bacteria can transfer plasmids back and forth allowing them to share these beneficial genes. This natural mechanism allows bacteria to adapt to new environments. The recent occurrence of bacterial resistance to antibiotics is due to the transmission of plasmids. Bio-Rad’s unique pGLO plasmid encodes the gene for GFP and a gene for resistance to the antibiotic ampicillin. pGLO also incorporates a special gene regulation system, which can be used to control expression of the fluorescent protein in transformed cells. The gene for GFP can be switched on in transformedcells by adding the sugar arabinose to the cells’ nutrient medium. Selection for cells that have been transformed with pGLO DNA is accomplished by growth on ampillicin plates. Transformed cells will appear white (wild-type phenotype) on plates not containing arabinose, and fluorescent green under UV light when arabinose is included in the nutrient agar medium. You will be provided with the tools and a protocol for performing genetic transformation. Your task will be to: 1. Do the genetic transformation. 2. Determine the degree of success in your efforts to genetically alter an organism.
STUDENT MANUAL LESSON 1
Lesson 1 Focus Questions There are many considerations that need to be thought through in the process of planning a scientific laboratory investigation. Below are a few for you to ponder as you take on the challenge of doing a genetic transformation. Since scientific laboratory investigations are designed to get information about a question, our first step might be to formulate a question for this investigation. 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 better suited for total genetic transformation— one composed of many cells, or one composed of a single cell?
2. 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?
3. 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?
4. Based on the above considerations, which would be the best choice for a genetic transformation: a bacterium, earthworm, fish, or mouse? Describe your reasoning.
STUDENT MANUAL LESSON 1
Consideration 2: How Can I Tell if Cells Have Been Genetically Transformed? Recall that the goal of genetic transformation is to change an organism’s traits, also known as their phenotype. Before any change in the phenotype of an organism can be detected, a thorough examination of its natural (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 following pre-transformation observations of E. coli
might provide baseline data to make reference to when attempting to determine if any genetic transformation has occurred. a) Number of colonies
b) Size of : 1) the largest colony 2) the smallest colony 3) the majority of colonies
c) Color of the colonies
d) Distribution of the colonies on the plate
e) Visible appearance when viewed with ultraviolet (UV) light
f) The ability of the cells to live and reproduce in the presence of an antibiotic such as ampicillin
1. 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.
2. What would you expect your experimental results to indicate about the effect of ampicillin on the E. coli cells?
STUDENT MANUAL LESSON 1
Consideration 3: The Genes Genetic transformation involves the insertion of some new DNA into the E. coli cells. In addition to one large chromosome, bacteria often contain one or more small circular pieces of DNA called plasmids. Plasmid DNA usually contains genes for more than one trait. Scientists use a process called genetic engineering to insert genes coding for new traits into a plasmid. In this case, the pGLO plasmid has been genetically engineered to carry the GFP gene which codes for the green fluorescent protein, GFP, and a gene ( bla ) that codes for a protein that gives the bacteria resistance to an antibiotic. The genetically engineered plasmid can then be used to genetically transform bacteria to give them this new trait.
Consideration 4: The Act of Transformation This transformation procedure involves three main steps. These steps are intended to introduce the plasmid DNA into the E. coli cells and provide an environment for the cells to express their newly acquired genes. To move the pGLO plasmid DNA through the cell membrane you will:
1. Use a transformation solution containing CaCl2 (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.
pGLO plasmid DNA
GFP
Flagellum
Pore
Cell wall
Beta-lactamase (antibiotic resistance)Bacterial chromosomal DNA
STUDENT MANUAL LESSON 1
Lesson 2 Transformation Laboratory Workstation (!) Checklist Your workstation: Materials and supplies that should be present at your workstation prior to beginning this lab are listed below.
Student workstation Material Quantity (!) E. coli starter plate 1 ” Poured agar plates (1 LB, 2 LB/amp, 1 LB/amp/ara) 4 ” Transformation solution 1 ” LB nutrient broth 1 ” Inoculation loops 7 (1 pk of 10) ” Pipets 5 ” Foam microcentrifuge tube holder/float 1 ” Container (such as foam cup) full of crushed ice (not cubed ice)1 ” Marking pen 1 ” Copy of Quick Guide 1 ” Microcentrifuge tubes 2 ”
Common workstation. A list of materials, supplies, and equipment that should be present at a common location to be accessed by your team is also listed below. Material Quantity Rehydrated pGLO plasmid 1 vial ” 42°C water bath and thermometer 1 ” UV Light 1 ” 37°C incubator 1 ” (optional, see General Laboratory Skills–Incubation) 2–20 µl adjustable volume micropipets 1 ” 2–20 µl micropipet tips 1 ”
STUDENT MANUAL LESSON 2
Transformation Procedure 1. Label one closed micro test tube +pGLO and another -pGLO. Label both tubes with your group’s name. 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.
+pGLO
+pGLO-pGLO
-pGLO
Transformation Solution
250 µl
STUDENT MANUAL LESSON 2
3. Place the tubes on ice.
4. Use a sterile loop to pick up 2–4 large colonies of bacteria from your starter plate. Select starter colonies that are “fat” (ie: 1–2 mm in diameter). It is important to take individual colonies (not a swab of bacteria from the dense portion of the plate), since the bacteria must be actively growing to achieve high transforation efficiency. Choose only bacterial colonies that are uniformly circular with smooth edges. 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 (with no floating chunks). Place the tube back in the tube rack in the ice. Using a new sterile loop, repeat for the -pGLO tube.
5. Examine the pGLO DNA solution with the UV lamp. Note your observations. Immerse a new sterile loop into the pGLO plasmid DNA stock tube. Withdraw a loopful. There should be a film of plasmid solution across the ring. This is similar to seeing a soapy film across a ring for blowing soap bubbles. Mix the loopful into the cell suspension of the +pGLO tube. Optionally, pipet 10 µl of pGLO plasmid into the +pGLO tube & mix. Do not add plasmid DNA to the -pGLO tube. Close both the +pGLO and -pGLO tubes and return them to the rack on ice.
+pGLO
Ice
(+pGLO)
pGLO Plasmid DNA (+pGLO) (-pGLO)
(-pGLO)
+pGLO
STUDENT MANUAL LESSON 2
6. Incubate the tubes on ice for 10 min. Make sure to push the tubes all the way down in the rack so the bottom of the tubes stick out and make contact with the ice.
7. While the tubes are sitting on ice, label your four LB nutrient agar plates on the bottom (not the lid) as follows:
• Label one LB/amp plate: + pGLO
• Label the LB/amp/ara plate: + pGLO • Label the other LB/amp plate: – pGLO
• Label the LB plate: – pGLO
8. Heat shock. Using the foam rack as a holder, transfer both the (+) pGLO and (-) pGLO tubes into the water bath, set at 42oC, for exactly 50 sec. Make sure to push the tubes all the way down in the rack so the bottom of the tubes stick out and make contact with the warm water. Double-check the temperature of the water bath with two thermometers to ensure accuracy. When the 50 sec are done, place both tubes back on ice. For the best transformation results, the transfer from the ice (0°C) to 42°C and then back to the ice must be rapid. Incubate tubes on ice for 2 min.
Water bath
L B /
a m p pGLO
L B / a m p / a r a pGLO
L B / a m p pGLO
L
B pGLO
Rack
Ice 42°C for 50 sec Ice
Ice
STUDENT MANUAL LESSON 2
9. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and, using a new sterile pipet, add 250 µl of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipet for the other tube. Incubate the tubes for 10 min at room temperature.
10.Gently flick the closed tubes with your finger to mix and resuspend the bacteria. Using a new sterile pipet for each tube, pipet 100 µl of the transformation and control suspensions onto the appropriate nutrient agar plates.
STUDENT MANUAL LESSON 2
11. Use a new sterile loop for each plate. Spread the suspensions 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. Uncover one plate at a time and re-cover immediately after spreading the suspension of cells. This will minimize contamination.
12.Stack up your plates and tape them together. Put your group name and class period on the bottom of the stack and place the stack of plates upside down in the 37°C incubator until the next day. The plates are inverted to prevent condensation on the lid which may drip onto the culture and interfere with your results.
STUDENT MANUAL LESSON 2
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 predictions.
2. If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be located? Explain your predictions.
3. Which plates should be compared to determine if any genetic transformation has occurred? Why?
4. What is meant by a control plate? What purpose does a control serve?
