TRANSFORMATION LAB
Introduction
In this lab, you will put a small circular piece of DNA, called a plasmid, into a bacterium called Escherichia coli (E. coli for short). This process, in which a new piece of DNA is placed into an organism, is called transformation. It is a part of the new "genetic engineering" technology. Transformation can be used to introduce useful new genes from other species into a species that normally doesn't have those genes. For example, plants and animals can be made resistant to certain diseases by this process. Some transformation experiments have produced interesting and strange results. In one famous case, the gene that makes a firefly glow is put into a plant. This results in a plant that can glow in the dark! On a more practical basis we are looking to do transformations within a species for gene therapy in such areas as cystic fibrosis and muscular dystrophy.
In this experiment, the bacterium E. coli cannot normally grow in the presence of the antbiotic ampicillin, nor can it break down the sugar-like molecule "X-gal". However, the DNA plasmid pUC19, which will be put into the bacteria, contains two important genes. The first gene codes for resistance to the antibiotic ampicillin, and the second gene codes for an enzyme which can beak down the X-gal. After undergoing transformation, the bacteria which successfully take in the new DNA will be identified by their ability to grow on ampicillin, and also by their ability to break down X-gal. Bacteria that can break down X-gal will turn a bright blue color, and so they will be especially easy to see.
Sterile Technique
If you are not careful, other bacteria and even a fungus or two may grow on your petri plates and contaminate your experiment. Do not leave the petri plate lids off any more than you have to. If you are using a sterile tool, such as a pipet or plastic loop, do not touch the tip or allow the tip to touch ANYTHING except the designated bacteria, agar or sterile surfaces. Never assume a surface is sterile unless it has been specially prepared. If you make a mistake and you think the tool is no longer sterile, don't take chances. Get another tool.
Objectives
* The student will define transformation.
* The student will transform a bacterium.
* The student will complete sterile technique.
* The student will interpret and analyze experimental results using comparisons with controls.
* The student will design an experiment using transformation technology.
* The student will calculate transformation efficiency.
Materials
1. Sterile pipet. Note the measurements on the diagram. It's a little hard to see the numbers on the actual pipet.
2. Sterile plastic "innoculating loop". The loop end must remain sterile. Touch this instrument only on the handle.
3. Culture E coli
4. pUC19 plasmid 10. test tube holder
5. Calcium Chloride 11. ice bucket
6. 2 plain Luria Broth agar petri plates 12. Luria Broth
7. 2 Ampicillin Luria Broth agar petri plates 13. Disposable gloves
8. 2 X-gal/Ampicillin Luria Broth agar petri plates 14. 42[[ring]] C water bath
9. 2 15 ml sterile culture tubes
Optional: Incubator
Procedure
1. First label one 15 ml test tube "+pUC" and the other "-pUC".
2. Use a sterile transfer pipet to add 250 ml of ice cold calcium chloride to each of the two tubes. If you then put the pipet carefully back into the package it came from, being careful not to touch the tip on anything but the inside of the package, it should stay sterile.
3. Place both test tubes in the ice bucket.
4. Use a sterile plastic loop to transfer some of the E. coli bacteria from the starter plate culture to the -pUC tube. Be careful not to scrape up any of the agar along with the bacteria; slide the loop smoothly over the surface to pick up the bacteria. You don't need tons of bacteria, but get enough so that you can see a small clump sticking to the loop. When you put the loop into the calcium chloride solution in the tube, tap it against the tube wall so that the bacteria fall into the calcium chloride. Store the loop carefully on the lab bench WITH THE TIP HANGING OVER THE EDGE SO IT DOESN'T TOUCH ANYHTHING. This will keep it sterile.
5. Now use the saved pipet to suck the suspension in and out a few times, so that the bacteria mix completely. No clumps of bacteria should be left in the tube. Place the pipette back into its wrapper.
6. Put the test tube back in the ice. Now take the +pUC tube and put some bacteria in it by following steps 4 and 5. You can use the same loop and the same pipet, as long as they have not touched anything which is not sterile.
7. Both tubes should now be on ice. Throw away the plastic loop AND the pipet.
8. Use a NEW sterile plastic loop to transfer a loopful of the pUC19 plasmid solution into the +pUC tube. Swish the loop in the tube to mix the DNA. Throw away the loop according to teacher instructions.
9. Return the +pUC tube to the ice. Leave both tubes on the ice for 15 minutes.
10. While the tubes are sitting on ice, label the six petri plates as follows:
Label one LB/Amp/X-gal plate "+" and the other one"-".
Label one LB/Amp plate "+" and the other one "-".
Label one LB plate "+" and the other one"-".
By the way, LB refers to the nutrient mixture that the bacteria grow on. Amp means the plate also contains the antibiotic ampicillin, and X-gal means it contains the sugar-like molecule X-gal.
11. Now that the test tubes have been on ice for 15 minutes, they need to be "heat shocked." This pulse will cause the cells to take up the DNA. To do this, remove the tubes from the ice and IMMEDIATELY put them in the 420 C water bath for one and a half minutes. Watch the time carefully; don't let them sit longer in the hot water.
12. Then IMMEDIATELY put the tubes back in ice for 1 minute (at least).
13. Use a NEW sterile transfer pipet to add 250 ml of LB broth to each tube. Put the pipet back in the package carefully to keep it sterile. Gently tap the tubes to mix them. Set the tubes in a test tube rack for a 10 minute recovery.
14. Now you are ready to put the bacteria on your labeled plates. To do this, use the same sterile transfer pipet to take 100 ml from the -pUC tube and put it on the - LB. Use the same pipet to add another 100 ml from the -pUC tube onto the -LB/Amp plate. Then use the same pipet again to add 100ml from the -pUC tube to the -LB/Amp/X-gal plate. Tilt lid over the plate only as much as necessary and replace it. Now all three of your plates labeled "-" should have 100 ml of -pUC suspension on them. Put the pipet carefully back in the package to store it.
15. Next you have to spread the bacteria over the surface of the plates. You can do this by quickly moving a new sterile loop back and forth across the plate surface. Be careful not to dig it into the agar. It should slide over the surface as if it were an ice skater. Use the same loop for all three plates and in the same that you have done so far (LB, then LB/Amp, then LB/Amp/X-gal).
16. Now use the sterile transfer pipet (the same one previously used as long as it has not touched the agar or any other object) to take100 ml of bacteria suspension from the +pUC tube and put it on the +LB plate. Then add 100 ml suspension to each of the other 2 plates (+LB/Amp and +LB/Amp/X-gal). You can use the same pipet for all three. Use the sterile loop to spread the suspension around on the three plates as described in step 15.
17. Tape the lids down on the plates (you can stack them all together). Put your name on the plates and place them UPSIDE DOWN in the 370 C incubator until the next day (or at room temperature for several days.)
Data
1. Observe, draw and describe each of the six plates carefully and write down observations for each one. How much bacterial growth do you see on each, relatively speaking? What color are the bacteria? You may be able to count how many bacterial colonies (the spots you see) that there are on each plate. This will depend on how many there are and how spread out they are.
Analysis
1. Why was one test tube labeled "+" and the other "-"?
2. Why do you suppose the CaCl2 (Calcium Chloride) was added to the test tubes?
3. Why did you add the same bacteria to each tube?
4. Why did you add pUC19 (the plasmid or ring of DNA) to the "+pUC" test tube ONLY?
5. Why did you heat shock the bacteria?
6. Why did you add LB broth to each test tube? (HINT: What is a broth?)
7. Why do you suppose you needed to let the bacteria sit in the test tube racks for another 10 minutes after heat shocking and adding broth?
8. For each of the plates listed below describe what the label means and give a prediction of growth:
Plate Description of Label Gowth/no Growth
"+LB"
"-LB"
"+LB/Amp"
"-LB/Amp"
"+LB/Amp/X-gal"
"-LB/Amp/X-gal"
9. Were you successful in transforming the bacteria? How do you know? Give possible explanations for ANY results that did not come out as expected. (Hint: review question #8 in procedure)
10. In this experiment, how many traits were placed into the bacteria? What are they and how are they expressed?
11. For each of the following pairs of plates, directly compare the results that you see. What does each pair of results tell you about the experimnent?
A and B
A and C
A and E
B and F
B and D
C and D
C and E
E and F
Conclusions
Describe how a transformation can be performed and methods in which to test the success of a transformation.
Extended Activities
I. Experimental Design Activity
1. Design an experiment where the technique of transformation is used to solve a problem for society. You may use different cells than E. coli or even a virus. You may also use different genes or plasmids. If necessary, you may use a different medium than LB broth with ampicilllin or X-gal. An example is that scientists have inserted normal genes into relatively undifferentiated cells of an individual with muscular distrophy. These are grown in a test tube and injected into the muscles of a person with the affliction. The transformation has remained effective only for a few months. Now design your own experiment using ideas from transformation technology. Make drawings if it enhances the description.
2. Analyze another person's or group's experiment. Are the steps, materials and equipment appropriate in their experimental design?
3. What kind of impact will this have on people, organisms, behavior and ecosystems? Including all positive and negative impacts, is the experimentation ethical, desirable and practical?
II. Determine Transformation Efficiency
By following the steps in this activity you will determine the number of antibiotic resistant colonies/ug pUC DNA.
1. Count the antibiotic-resistant colonies in +LB/Amp/X-gal plate.
# =
2. Mass = Concentration X Volume Step 8
ug = ug/ul X ul of pUC
3. Fraction spread = Volume suspension spread/Total volume
/ = ul / ul
Fraction of cell suspension Suspension Total volume for
spread on LB/Amp/X-gal spread on top entire test tube
plate of plate before withdrawn
(CaCl & LB broth)
Steps 14,15,16 pUC & E.coli is
too minimal to
worry about
4. Mass pUC Spread = Total Mass plasmid X Fraction Spread
ug = ug X /
Computation 2 Computation 3
5. Transformation Efficiency = #colonies observed/mg pUC
= / ug
Count Computation4
6. Do steps 1-5 for the +LB/Amp plate.
a) Count =
b) Mass = X = ug of pUC
c) Fraction spread = /
d) Mass pUC Spread = ug
e) Transformation Efficiency = transformants/ul
7. What factors might influence transformation efficiency?