Fluorescent Protein Transformation Student Background

TRANSFORMATION LAB

Genetic transformation occurs when a cell takes up (i.e. takes inside) and expresses a new piece of genetic material—DNA. Genetic transformation literally means change caused by genes and it involves the insertion of a gene(s) into an organism in order to change the organism’s traits. Remember that a gene is a piece of DNA which provides the instructions for making (coding for) a particular protein.

Genetic transformation is used in many areas of biotechnology. In agriculture, genes coding for traits such as frost, pest or drought 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 gene involved in their disease. Another medical application is in the creation of proteins, such as insulin (synthesized by Genentech) and factor VIII (blood clotting protein synthesized by Bayer). Genes can be cut out of human, animal or plant DNA and placed inside bacteria. For example, a healthy human gene for the hormone insulin can be put into bacteria. Under the right conditions, these bacteria can make authentic human insulin just as they would make their own proteins. This insulin can then be used to treat patients with the genetic disease, Diabetes, whose insulin genes do not function properly.

amp

Rfp

In this lab, you will learn about the process of moving genes form one organism to the other with the aid of a plasmid. In addition to one large circular chromosome which contains all of the genes a bacterium needs for its normal existence, bacteria naturally contain one or more tiny circular pieces of DNA called plasmids. Plasmid DNA contains genes for traits that may be beneficial to bacterial survival under certain environmental conditions.

In nature, bacteria can transfer plasmids back and forth, allowing them to share these beneficial genes. This mechanism allows bacteria to adapt to new environments. The recent occurrence of bacterial resistance to antibiotics is due to transmission of plasmids. In order to do transformation, the gene to be transferred is placed into a plasmid. This is done with the help of restriction enzymes, naturally occurring enzymes from bacteria that recognize a particular sequence of DNA bases and cut at that sequence. Bacteria use restriction enzymes to protect themselves from viruses which inject their DNA into the bacteria; the enzymes can cut the viral DNA before it can hurt the bacteria. The same restriction enzyme is used to cut the ends of the gene we want to transfer and to cut open the plasmid. Because the cuts are made at the same base sequence, the ends will match and reattach when placed together. The plasmid used in this lab is called pARA-R developed by Amgen Laboratories for use in the classroom. The plasmid is used as the vector (transport mechanism) that will transform a nonpathogenic (non-disease causing) E. coli bacteria.

The pARA-R plasmid contains a gene for the Red Fluorescent Protein (RFP) that comes from the bioluminescent from a sea anemone, a soft-bodied animal related to coral and jellyfish. This gene allows the sea anemone to produce a protein that fluoresces, or glows, under ultraviolet light. On the right is a picture of the molecular structure of one fluorescent protein (Red Fluorescent Protein or RFP). You can see it has an interesting barrel shape. The part of the molecule that actually fluoresces, the chromophore, is in the middle of the “barrel” and is composed of a ring of only 3 amino acids and believe it or not the organism doesn’t need to use energy to make the protein light up; the protein is simply “energized” by light hitting the chromophore.

The gene can be turned on in transformed cells by simply adding the sugar, arabinose, to the cells’ nutrient medium. Without arabinose, the bacteria will not fluoresce. So arabinose is the “switch” for the RFP gene in this plasmid. To move the plasmid through the E. coli cell membrane you will use a transformation solution of calcium chloride (CaCl2), and a procedure know as “heat shock” which will open small pores in the bacteria.

In order to select only the bacteria that have received the new gene, the plasmid also has a gene for resistance to the antibiotic Ampicillin.

The gene codes for the production of a protein, betalactamase, that allows the bacteria to digest the antibiotic before it can cause any harm. Therefore, if ampicillin is mixed into the agar on the bacterial plates, the only bacteria that can survive there will be bacteria with the gene/plasmid for ampicillin resistance. The ampicillin resistance gene inserted into the plasmid allows scientists to select bacteria that have been transformed, and for that reason it is called a selectable marker. Since we know that the gene for Red Fluorescent Protein is on the same plasmid, if any bacteria grow on the plate, they must have been transformed, even if we can’t see them glow. Transformed cells will appear white and grow in circular colonies. If arabinose is present, these colonies will appear bright pink!

Below is a map of the plasmid used in this lab. By examining the growth of bacteria under these conditions, you can verify that your procedure worked, and you can identify the bacteria transformed with the plasmid. How will you know if you are successful? The bacteria will have a new and highly visible trait: It will now produce red fluorescent protein, which makes the cells red or bright pink!

Hind III

BamHI

Hind III

BamHI

With the addition of the restriction enzymes and ligase steps we will add the RFP gene to make

BamHI

Hind III

Plasmid to be inserted

BACTERIAL GROWTH PREDICTIONS

10. Predict how much bacterial growth you will see on each plate. Mark the plate/plate section with +++ (for high growth), ++ (for medium growth), + (low growth), or - (for no growth):

Table 1: P- Control Group (Non- Transformed Bacteria)

Plate Contains:

Predicted Growth

Conclusion if Predicted Growth occurs

Conclusion if Predicted Growth Does Not Occur

Luria Broth

(LB)

Luria Broth

Ampicillin

(LB/amp)

Table 2: P+ Experimental Group (Transformed Bacteria)

Plate Contains:

Predicted Growth

Conclusion if Predicted Growth occurs

Conclusion if Predicted Growth Does Not Occur

Luria Broth

(LB)

Plasmid

Luria Broth

Ampicillin

(LB/amp)

Plasmid

Luria Broth ampicillin arabinose

(LB/amp/ara)

Plasmid

Student Transformation Lab Protocol Period_____

Materials

·

The following will be given by teacher:

· plasmid with rfp gene (RP)

· Luria Broth (LB)

· Competent E. coli cells (CC)

· P-20 micropipette

· P-200 micropipette

· Tip box of disposable pipette tips

· 42°C water bath

· 37°C incubator

Checklist of materials at each lab station:

· 3 Petri plates with agar:

· 1 of LB

· 1 of LB/amp

· 1 of LB/amp/ara

· 2 1.5-mL microfuge tubes

· Permanent marker

· Styrofoam cup of Crushed ice

· Pack of cell spreaders

· Timer

· 2 Transfer pipettes (sterile)

· Waste Container 10% bleach

Note: Fill a cup with some of the crushed ice with a little water. You’ll need to keep the CC tube on ice at all times.

Checklist for Procedure:

1. Check to be sure you have all the needed materials at your lab station.

2. Label two clean microfuge tubes “P–” and “P+” along with your initials and class period.

3. Place the P– and P+ and tubes in the Styrofoam cup of ice.

LAB TECHNIQUE: Bacterial transformation requires sterile techniques. Follow all directions precisely.

4. With the help of your teacher, add 50 μL competent cells (CC) from the CC tube to the P– and P+ tubes:

5. Holding each tube at its rim to keep it cold, and return each tube quickly to the ice.

LAB TECHNIQUE: To avoid contamination, be sure to use a new micropipette tip for each addition.

6. With the help of your teacher add 10.0 μL of the RP (Red Plasmid) to the tube labeled “P+”:

7. Keep the P– and P+ tubes on ice for 15 minutes.

NOTE: During the 15-minute interval, do the following steps.

8. While the cells are on ice, prepare your three agar Petri plates—one plate each of LB has one stripe, LB/amp has two stripes, and LB/amp/ara has three stripes:

A. Label the bottom of each plate (the part that contains the agar) with your group number and class period. Write small and on the edge of the plate.

9. With the plates closed, draw a line on the LB plate and the LB/amp plate that divides each plate in the middle. Label half of each plate “P–” and the other half “P+.” Label the LB/amp/ara plate “P+.”

The plates will be arranged as follows:

Following the 15 minute interval.

10. Heat shock: Take your ice water bath with its tubes to the 42oC hot water bath. Transfer the tubes to the hot water and time for exactly 45 seconds.

A. Make sure that the tubes are in contact with the hot water.

B. Immediately return the tube to the ice and leave them there for at least a minute.

11. Using a transfer pipette, add 250 μL LB to the P– and P+ tubes. The line below the .5mL mark

12. If time permits, allow the cells in the P– and P+ tubes to incubate at room temperature for 15 minutes.

13. Open the P- tube, and with a clean pipette, gently pump the pipette two or three times in the P– tube to suspend the cells.

A. Transfer about half of the cell mixture to the P- side of LB petri dish

B. Add the rest of this tube to the P- side of LB/amp petri dish.

C. Do not discard the pipette.

14. With the same pipette, gently pump the pipette two or three times in the P+ tube to suspend the cells.

15. Transfer half of the mixture from the P+ tube to the LB/amp/ara P+ plate.

16. The remainder of the mixture be split between the LB P+ plate, LB/amp P+ plate.

17. Place the pipettes in the waste container.

LAB TECHNIQUE: Hold the spreader by the handle and do not allow the bent end to touch any surface, as this will contaminate the spreader.

18. Spread the cells on each plate with a clean, sterile spreader, this will help to distribute the liquid evenly.

A. The spreader should glide along the surface of the agar, do not dig into the plate

B. Makes sure not to cross over the black line with the spreader.

C. Let this sit for a few minutes to absorb the liquid.

19. Place the used spreader in the bleach at the teacher station

20. Using provided tape, tape all three plates together and label tape with your group number and class period.

21. Place the plates in the 37°C incubator upside down to prevent condensation from dripping onto the gels.

22. Place all microtubes, pipette tips, in the bleach

23. Incubate the plates for 24–36 hours at 37°C.

24. Examine the plates and in your notebook record the amount of growth on each half.

25. Discard the Petri plates in the bleach when directed to do so.

Name ___________________________________________________________Per________

PRE-LAB QUESTIONS: All answers must be in complete sentences

1. What does the term transformation mean?

2. What bacteria are we using in this lab?

3. List and describe at least 3 things that scientists use transformation technology for.

4. What is the purpose of adding ampicillin the agar plates?

5. What is the purpose of the calcium chloride (CaCl2) and heat shock?

6. How will you know if your bacteria have been transformed? Be Specific!

7. What does it mean to fluoresce?

8. Why do plasmids make good vectors (something that transfers DNA from one place to another)?

9. Summarize the procedure steps in your own words (use sketches or cartoons if necessary)

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POST LAB WRAP-UP QUESTIONS: All answers must be in complete sentences

1. Draw and Color what your plates look like after 24-48 hours

2. What is a plasmid?

3. Why are plasmids used for transformation?

4. What genes does the pARA-R plasmid carry?

5. How are restriction enzymes used in this lab?

6. What is the natural function of restriction enzymes in bacteria?

7. Why is it important that we use the same restriction enzyme on the plasmid and the florescence gene?

8. What protein does the gene for antibiotic resistance code for?

9. What does the antibiotic resistance gene allow the bacteria to do to the antibiotic? Be specific!

10. What is the purpose of florescent proteins in nature?

11. What is a chromophore?

a. How many amino acids is it made of?

b. Does the cell use energy to “glow”

12. What is special about florescent proteins that allow them to be used for medical research?

13. How is a selectable marker used in this lab?

14. How many red colonies were present on your LB/amp/ara plate?

15. Why did the red colonies only appear on the LB/amp/ara plate and not the LB/amp plate?

16. What do your experimental results to indicate about the effect ampicillin has on E. coli cells?

17. Based on your results what molecule is required to turn on the glow gene?

18. Look at the results of your transformation. Do your actual results match your predicted results? If not, what differences do you see, and what are some explanations for these differences?

19. Suppose that a group did not get any transformed colonies. What errors could have caused this?

20. Recombinant plasmids are engineered so that they can replicate in the cell independently of the chromosome replication. Why is it important to have multiple copies of a recombinant plasmid within a cell?

21. Describe how a strain of bacteria could develop antibiotic resistance.

a. Why is this a problem

22. The RFP gene on DNA is 702 nitrogen bases long, use what you have previously learned about gene expression and the relationship between DNA, RNA, proteins to show how the start of this protein was made.

DNA: TAC AAG CTT GCA TGC CTG CAG

mRNA:

tRNA:

AA:

23. Google the various uses of Florescent Proteins and summarize what you found in (provide the source)

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