Introduction

Description

This exercise demonstrates the use of competent E. coli cells in the take up of plasmids to cause their transformation. The strain used in this exercise is JM83 and they may be acquired competent from UC Davis or the cells could be made competent in a previous exercise. These cells are made to take up pUC19, which contains two engineered genes, one for ampicillin resistance (ampicillin resistance gene) and the other (ß-galactocidase gene) to convert X-gal in nutrient agar to a blue color. 

A sample of competent cells is made to take up pUC19 with the two genes. When E. coli with this insert is plated on nutrient agar with ampicillin and X-gal, the resulting colonies will be blue. Another sample is made to take up another pUC19 with a DNA insert (recombinant pUC19). The DNA is inserted in a restriction site located within the ß-galactocidase gene. When E. coli with the recombinant pUC19 are plated out on ampicillin nutrient agar with X-gal, the gene that would normally convert X-gal to a blue color is no longer functioning because the DNA insert disrupted the gene. These colonies will be white (the ampicillin gene is not affected).

This experiment demonstrates that DNA can be inserted into bacteria to change the properties of the cells. This exercise also demonstrates that colonies can be screened to determine which ones took up a plasmid, and of those that did, which ones contain the recombinant plasmid with the DNA insert.

Student Audience

This is easily done at the college level introductory majors' biology level. However, several local high school instructors use similar recombinant experiments in their biology course.

Goals for the Experiment

• Learn sterile technique
• Understand the properties of DNA and genes as it applies to transformation
• Screen for those bacterial with inserted DNA for future experiments
• Understand the importance of plasmids as a vector to transfer genes of interest

This exercise works well after the student has been introduced to the basic principles of DNA and is now ready to learn how this information can be applied in biotechnology. The field of biotechnology and recombinant DNA can then be discussed.
 

Purpose

The purpose of this exercise is to:

• Gain a working knowledge of recombinant techniques, 
• Understand the principles of screening for a gene of interest, 
• Understand the function of genes and that genes are found on the DNA molecule,
• DNA can be transferred to another organism and therefore change the phenotype of that organism.

You have learned that genes are composed of DNA that contains the code to determine the phenotype of an organism. In this way an organism can produce the necessary enzymes to carry out the specific functions which allow it to live and reproduce in its environment.

In this experiment we will be using E. coli because it is a fairly easy organism to grow and observe. The strain of E. coli being used is JM83. This strain has been made competent to take up a small circular DNA called a plasmid. On this plasmid are certain genes that give E. coli the ability to perform certain functions not present in its genome (library of genes). When E. coli has received this "new" set of genes it is said to be transformed.

The plasmid being used is called pUC19. This plasmid has been engineered with two genes useful in transformation experiments. One gene (AP^r) confers resistance to the antibiotic ampicillin, and the other gene (B-galactosidase gene derivative) produces a product that can convert a colorless compound X-gal into a blue derivative that colors the bacterial colonies blue.

It is interesting to note that pUC19 has many sites within the B-galactosidase gene where DNA from another source can be inserted (multiple cloning site or MCS). However, if foreign DNA is inserted within this gene, the gene no longer has the ability to function properly and it is not able to produce a product that converts X-gal into a blue derivative which in turn results in white colonies. The ampicillin resistance gene in not affected. Therefore, when we see white colonies growing on ampicillin media, we can assume that these bacteria have taken up the recombinant pUC19 (pUC19 with the DNA insert).

E. coli is sensitive to ampicillin and will not grow on nutrient plates in the presence of this antibiotic. E. coli with pUC19 is resistant to ampicillin and will grow on these plates (this shows that DNA equals genes or traits). E. coli with pUC19 also will be seen as blue colonies. Only the recombinants (those that took up pUC19 with a DNA insert) will be seen as white colonies (changing DNA changes the properties of the genes). In this way we can distinguish between E. coli with the original wild-type pUC19 and E. coli with the recombinant pUC19, which contains the DNA insert. We could, if we wanted to, pick up the recombinant E. coli to grow separately from the other colonies. 
 

Scenario/Industrial Applications

Similar techniques are used to create a library of the genome of a particular organism. These techniques can also be used in agriculture and industry to create new organisms with specific adaptations required and to produce products used in medicine and pharmaceuticals.

Questions:

What product would we expect to see if we selected the recombinant colony that had an inserted human insulin gene in its pUC19? 
 
 

What color would this colony be on X-gal medium?
 
 
 
 

Would this same colony also have resistance to ampicillin? Why?
 
 
 
 

What would be a specific agricultural application of recombination?
 
 
 
 

What would be a specific industrial application of recombination?
 
 
 
 
 

Safety, Handling, and Disposal

Sterile technique must be practiced at all times to prevent contamination of the experiment and to prevent release of organisms and/or chemicals in the lab. Other students use the lab and we do not want anything to leave the lab that could be dangerous to others or the environment. Wear gloves when doing this exercise and dispose of used reagents, gloves, and other material according to local ordinances. Pay attention to notices instructing you on the disposal of all items. When in doubt, ask!

Procedure: The entire procedure must be done with sterile technique. Review and practice this technique before actually doing the lab experiment.

Materials Needed

Reagents: (per group)

Two 0.5 ml tubes of Competent E. coli (200 µl of JM83) in 0.5 ml tube

One 0.5 ml tube of PUC19 (10 µl of 100 µg/ml)

One 0.5 ml tube of recombinant pUC19 (10 µl of 100 µg/ml)

One nutrient agar plate

Five nutrient agar plates containing 100 µg/ml ampicillin (NA+Ap plates)

Tube of 2% X-gal in dimethylformamide (this solvent is necessary for X-gal)

Tube of 100 mM IPTG in sterile water

Ethanol 95% in beaker (for sterilizing spreader bar)
 
 

Equipment and Supplies:

A water bath or temperature block at 43°C.

Incubator for plate culture (may be done at room temperature, but will take two days)

Glass or stainless steel spreader bar

P-20 micropipette and yellow tips

P-200 micropipette and yellow tips

P-1000 micropipette and blue tips

0.5 ml tubes

Crushed ice

Bunsen or propane burner

Waste container with 10% bleach solution
 

Instructions for Students:
 

1. Pick up from the front counter:

• one nutrient agar plate 
• five nutrient agar plates with ampicillin 
• two small tubes of JM83 competent cells (JM83 200 µl)
• two small empty sterile tubes
• one small tube labeled "pUC19 DNA"
• one small tube labeled "pUC19 Recombinant"
• small tube of X-gal/IPTG mix (300 µl)
• one spreader
• beaker of ethanol
• beaker of ice

2. Place 200 µl of the X-gal in a plastic tube. Add 50 µl of IPTG to the tube and mix the two solutions.

3. Transfer 60 ul of the X-gal/IPTG mixture on the surface of one of the ampicillin plates. Dip a glass spreader in 95% ethanol and flame to sterilize and let it cool for a few seconds by touching the agar surface. Use the spreader to distribute the X-gal/IPTG mixture evenly over the surface of the plate. Replace the lid on the plate and invert the plate. Repeat this process for three other plates of ampicillin. You should now have four plates of X-gal/IPTG and ampicillin (label them X-gal).

3. Take one of the tubes of competent cells and label it "no DNA". Remove 100 µl of the cells from this tube and place in a sterile tube. Label this tube "no DNA, Ap). Return these tubes to the ice.

4. Take the second tube of competent cells and label it "pUC19". Remove 100 µl of cells from this tube and place in a sterile tube labeled "Recombinant". Return tubes to ice.

5. Place 2 µl of pUC19 DNA in the tube of competent cells labeled "pUC19". Return the tubes to ice.

6. Place 2 µl of the recombinant plasmid DNA in the tube of competent cells labeled "Recombinant". Return the competent cells to the ice.
 

• You will now have four tubes of competent cells:

• No DNA
• No DNA, Ap
• pUC19
• Recombinant

8. To make the DNA enter the cells, place all four tubes in a 43oC water bath. Allow the tubes to incubate at 43oC for 60 seconds, then remove them from the water bath and place them at room temperature.

Now it's time to transfer the JM83 cells to selective agar plates to allow them to grow and demonstrate the results of our experiment.

9. Transfer the contents (about 100 µl) of tube 1 "no DNA" to the nutrient agar (NA) plate. Use the spreader to distribute the cells. Label the plate "NA no DNA". 

10. Transfer the contents (about 100 µl) of tube 2 "no DNA, Ap" to the nutrient agar/ampicillin plate (NA+Ap). Use the sterile spreader to distribute the cells. Label the plate "NA+Ap no DNA".

11. Transfer 10 µl of cells from tube 3 "pUC19" to one X-gal plate (NA+Ap+X-gal/IPTG) and the rest of the cells (about 90 µl) to another X-gal plate. Use the sterile spreader to distribute the cells. Label the plates "NA+Ap 10 ul pUC19" and "NA+Ap 90 ul pUC19".

12. Transfer 10 ul of the cells from tube 4 "recombinant" to one X-gal plate and the rest of the cells (90 µl) to another X-gal plate. Remember that each X-gal plate also had nutrient agar and ampicillin. Use the sterile spreader to distribute the cells. Label the plates "NA+Ap 10 µl recombinant" and "NA+Ap 90 µl recombinant". 

13. Incubate the plates at 37oC for 12 to 18 hours to allow growth of colonies. (We will incubate these plates for you and provide them for you to read during the lab period next week).

14. Write down any questions you may have about this lab and bring them up in lecture or the following lab.

You should obtain the following pattern:

The "no DNA" nutrient agar plate demonstrates that the cells are viable and can grow in the absence of ampicillin. The "no DNA" ampicillin plate demonstrates that the cells cannot grow in the presence of ampicillin because they do not contain the necessary DNA (gene) to allow them to do so.

Transformation with pUC19 confers the ability to grow in the presence of ampicillin and produce products to convert X-gal to produce blue colonies. The addition of an extra DNA fragment in the recombinant "pUC19 recombinant" does not affect the ability of the recombinant to be ampicillin resistant and therefore grow in ampicillin agar. However, the colonies remain white because when the extra DNA fragment was inserted it inactivated the X-gal gene. The bacteria were not able to use that gene so no blue color resulted.
 
 

This exercise demonstrates two fundamental principles of recombinant DNA methods:

• DNA can be inserted into bacteria to change the properties of the cells (ampicillin-resistant colonies).

• DNA fragments can be re-arranged to change the genetic properties of the DNA molecules (blue colonies caused by pUC19 are converted to white colonies when DNA fragments are inserted into pUC19 DNA).

Questions:

1. Do your plates follow the predicted growth pattern? Explain any variation from the predicted growth pattern.
   
   
2. Why was there no growth in the "No DNA" bacteria spread in the nutrient agar with ampicillin? What does this demonstrate?

 
3. Why were there colonies and not lawn in the plates with ampicillin?

 
4. If you should have obtained white colonies in the "recombinant" plate, what would account for some blue colonies?
   
   
5. Why was it necessary to re-flame the spreader between each inoculation?

Micklos, David A. and Freyer, Greg A. DNA Science. A First Course in Recombinant DNA Technology. Cold Spring Harbor Laboratory Press. 1990

Bloom, Mark V., Freyer, Greg A., and Micklos, David A. Laboratory DNA Science. The Benjamin/Cummings Publishing Company, Inc. 1996

Title:

Transformation of Competent Cells with a Recombinant Plasmid

Time Required:

About 90 minutes not counting the incubation time and read out at the next lab/class meeting.

Group Size:

Two to Four students (ideally three: one to read instructions, one to perform manipulations, one to record).

Material Needed:

Reagents: (per group)

Two 0.5 ml tubes of Competent E. coli (200 µl of JM83) in 0.5 ml tube

One 0.5 ml tube of PUC19 (10 µl of 100 µg/ml)

One 0.5 ml tube of recombinant pUC19 (10 µl of 100 µg/ml)

One nutrient agar plate

Five nutrient agar plates containing 100 µg/ml ampicillin (NA+Ap plates)

Tube of 2% X-gal in dimethylformamide (this solvent is necessary for X-gal)

Tube of 100 mM IPTG in sterile water

Ethanol 95% in beaker (for sterilizing spreader bar)
 
 

Equipment and Supplies:

A water bath or temperature block at 43 degree Celsius.

Incubator for plate culture (may be done at room temperature, but will take two days)

Glass or stainless steel spreader bar

P-20 micropipette and yellow tips

P-200 micropipette and yellow tips

P-1000 micropipette and blue tips

0.5 ml tubes

Crushed ice

Bunsen or propane burner

Waste container with 10% bleach solution
 
 

Safety, Handling, and Disposal:

Sterile technique must be practiced at all times to prevent contamination of the experiment and to prevent release of organisms and/or chemicals in the lab. Wear gloves when doing this exercise. Be careful to instruct students in the safe use of flammables such as 95% ethanol. Keep the open beaker of ethanol a safe distance away from the flame of the Bunsen burner (or propane unit) and out of the way so it does not get bumped over. Remove the beaker of ethanol immediately once the spreading procedure is accomplished. Dispose of used reagents, gloves, and other material according to local ordinances.

Points to Cover in the Pre-Lab Discussion:

• Discuss the general concept behind recombination and bacteria including DNA, genes, plasmids, and antibiotics. 
• Mention the importance of sterile technique and give brief instructions on how to use sterile technique. 
• Remind the students to read the procedures before (not only during) the actual experiment. Have them decide what responsibilities are to be assigned to each group member. 
• Stress the importance of following the times for the heat/cold treatment for the take up of the plasmid. 
• Instruct the student to remove gloves when leaving the laboratory for a break, and replacing gloves when returning.
• Review disposal procedures in accordance with local ordinances.

• If more time permits, the steps to make the cells competent could be included in the same lab (especially if it may be a three-hour lab). The students were given competent cells in the same laboratory period just to save time, especially if other procedures are planned during the same lab period. Competent cells should be used within the week or stored at -70?C, otherwise they will slowly lose the ability to take up plasmid.
• Ampicillin may be purchased separately and added to cooled agar before it is poured or it may be added to the cell solution. 
• Purchasing plates is convenient and saves time but is of course more expensive.
• An in expensive pipette may be made using 1 ml syringe and yellow plastic tips. The yellow tips would need to be cut with a razor blade near its base so that it will fit snuggly on the syringe tip. The gradations on the side of the syringe are usually in 0.01 ml (10 µl). These could be used to deliver cell mixtures or X-gal/IPTG mixes to the agar plates.

Plausible Answers to Student Questions:

After Introductory Comments:
 

What color would this colony be on X-gal medium?

White

Would this same colony also have resistance to ampicillin? Why?

Yes, because it contains the plasmid with the ampicillin gene still intact.
 

What would be a specific agricultural application of recombination?

Making bacteria with genes to prevent strawberries from freezing. One could also use another vector and cause root cells in a plant to take up the gene for nitrogen fixation, and therefore reduce the need for as much fertilizers.

What would be a specific industrial application of recombination?

Using E. coli with a plasmid with the human insulin insert to produce insulin for diabetic patients.
 

After Reading Results of Exercise:

6. Do your plates follow the predicted growth pattern? Explain any variation from the predicted growth pattern.

 
(Open-ended question, would vary according to results)

7. Why was there no growth in the "No DNA" bacteria spread in the nutrient agar with ampicillin? What does this demonstrate?
 

These bacteria did not have the plasmid with ampicillin resistance. This demonstrates that the original bacteria used in this experiment do not contain the gene for ampicillin resistance. This is a control.

8. Why were there colonies and not lawn in the plates with ampicillin?
 
 

Not all of the bacteria were able to take up a plasmid. There is an efficiency quotient depending on the strain, method, and type of plasmid. Those that did not take up the plasmid could not grow in ampicillin agar. The few that did take up the plasmid each formed a colony.
 

9. If you should have obtained white colonies in the "recombinant" plate, what would account for some blue colonies?
 

Not all of the plasmids actually took up the DNA insert in the procedure used to produce the recombinant plasmids (restriction/ligation with specific restriction enzymes and DNA). Some bacteria could have taken up those plasmids that did not have DNA insert and therefore had a ß-galactosidase gene that was not disrupted would function normally to convert the X-gal/IPTG to a blue color.
 
 

10. Why was it necessary to re-flame the spreader between each inoculation?
 

So as to not contaminate a plate with the bacteria that was spread in the previous plate. If that should happen, there may be several "experiments" in the same plate, and the results would not be accurate.

Extensions and Variations:

• This lab could be taken a step further by having the students produce their own competent cells to begin with. Also, a colony could be picked off the plate, cultured, and the plasmid purified. 
• Electrophoresis could be done to analyze the fragment size of the plasmid with the DNA insert (white colony), compared to a plasmid from a colony that did not have the inserted DNA (blue colony).

References:

Bloom, Mark V., Freyer, Greg A., and Micklos, David A. Laboratory DNA Science. The Benjamin/Cummings Publishing Company, Inc. 1996.

Brown, T. A. Essential Molecular Biology. A Practical Approach. Volume 1. Oxford University Press. 1991.

Micklos, David A. and Freyer, Greg A. DNA Science. A First Course in Recombinant DNA Technology. Cold Spring Harbor Laboratory Press. 1990. 

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