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Green Fluorescent Protein Lab

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Category: Science

Autor: reviewessays 18 December 2010

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Title: Purification of Green Fluorescent Protein

Introduction: Transformation is used to introduce a gene coding for a foreign protein into bacteria. Hydrophobic Interaction Chromatography (HIC) is used to purify the foreign protein. Protein gel electrophoresis is used to check and analyze the pure protein. Research scientists use Green Fluorescent Protein (GFP) as a master or tag to learn about the biology of individual cells and multicultural organisms. This lab introduces a rapid method to purify recombinant GFP using HIC. Once the protein is purified, it may be analyzed using polysaccharide gel electrophoresis (PAGE).

Purpose: To illustrate the process of transformation and perform it using Green Fluorescent Protein.

Experimental Design: This experiment will achieve its purpose by allowing the student to perform a transformation following step by step instructions.

Procedure:

Laboratory Procedure for pGREEN Steps

1. Mark the sterile 15mL tubes with the correct labelings. (LB+plasmid, LB/Amp+plasmid).

2. Use a sterile transfer pipet to add 250mL of ice cold calcium chloride to each tube.

3. Place both tubes on ice.

4. Use a sterile plastic inoculating tube to transfer isolated colonies of E.coli from the starter plate to the +plasmid tube.

5. Immediately suspend the cells by repeatedly pipetting in and out with a sterile transfer

pipet. Examine the tube against light to confirm that no visible clumps of cells

remain in the tube or are lost in the bulb of the transfer pipet. The suspension

should appear milky white.

6. Return the +plasmid tube to ice.

7. Use a sterile plastic inoculating tube to add one loopful of DNA to the +plasmid tube.

immerse the loopful of plasmid DNA directly into the cell suspension and spin the

loop to mix the DNA with the cells.

8. Return the +plasmid tube to ice and incubate for 15 minutes.

9. While the tubes are incubating, label your media plates.

10. Following the 15 minute incubation on ice, heat shock the cells using a water bath.

11. Use a sterile transfer pipet to add 250 uL Luria broth to each tube. Gently tap the

tubes to mix the LB with the cell suspension. Place the tubes in a test tube rack for a 5 to 15 minutes recovery.

12. Now you will remove some cells from each transformation tube and spread them on the plates.

13. Use a sterile transfer pipet to add 100uL of cells from the -plasmid transformation to the appropriate plate.

A. clam shell the lids and carefully pour 4-6 glass beads per plate

B. use a back and forth shaking motion to move the glass beads

C. let the plates rest for several minutes

D. remove the glass beads

15. Transfer 100uL of cell suspension from the +plasmid tube to each appropriate plate

16. Immediately suspend the cells

17. Wrap the plates together with tape and place the plates upside down in the incubator.

Purification of GFP by HIC Steps

1. Shake the culture tube to suspend the E.coli cells.

2. Use a micropipette to transfer 1mL of the overnight E.coli/GFP culture into a 1.5mL

tube.

3. Cap the tube and place it in a balanced configuration in the micro centrifuge rotor. Spin for one minute to pellet the cells.

4. Carefully pour off the supernatant. Do not disturb the green cell pellet.

5. Repeat steps 1-4 in the same 1.5 mL tube to pellet cells from a second 1 mL sample on

top of the first pellet. This will result in a large, green cell pellet.

6. Add 500mL of lyses buffer to the tube. Resuspend the cell.

7. Incubate the tube on ice for 15 minutes.

8. Microcentrifuge for 5 minutes.

9. Transfer 250mL of green cell extract into a clean 1.5mL tube.

10. Add 250mL of binding buffer to the tube containing 250mL of cell extract. Invert

11. Add 400mL of the cell extract/ binding buffer mixture to the tube of hydrophobic bead resin. Invert.

12. Microcentrifuge for 30 seconds.

13. Add 400mL of wash buffer to the hydrophobic bead pellet. Invert.

14. Microcentrifuge for 30 seconds.

15. Elute the recombinant GFP by adding 200mL of TE buffer to the hydrophobic bead

pellet. Invert.

16. Microcentrifuge for 1 minute. Use a micropipette to transfer the supernatant containing the recombinant GFP to a new 1.5mL Eppendorf tube.

17. Observe the GFP under ultraviolet light.

Data: (Laboratory Procedure for pGREEN)

1./2./3.

LB-plasmid: LB+plasmid: LB/Amp+plasmid: LB/Amp-plasmid

Prediction: average growth average growth growth no growth

Reason: control control it has LB/Amp and plasmid no plasmid

Result: lawn lawn 17 colonies no growth

4.

LB+plasmid (Pos Control) lawn LB-plasmid (Pos Control) lawn

LB/Amp+plasmid (Exper) 17 colonies LB/Amp-plasmid (Neg Control) no growth

5.

a. LB+plasmid and LB-plasmid: Both of these plates had a lawn of bacteria. This proves that the bacteria are capable of growing on the agar and that there was nothing preventing growth beside the ampicillin.

b. LB/Amp-plasmid and LB-plasmid: The LB/Amp-plasmid had no growth compared to the LB-plasmid which had a lawn. This proves that transformation cannot take place without the plasmid, or DNA.

c. LB/Amp+plasmid and LB/Amp-plasmid: The LB/Amp-plasmid had no growth, but the LB/Amp+plasmid had growth. This shows that the bacteria was transformed and developed a resistance to ampicillin.

d. LB/Amp+plasmid and LB+plasmid: The LB/Amp+plasmid had less growth than the LB+plasmid. This shows that the transformation was not completely effective and only transformed some of the most competent bacterial cells.

Analysis:

Laboratory Procedure for pGREEN Questions

6. What are you selecting for in this experiment? (i.e., what allows you to identify which bacteria have taken up the plasmid??

The AMP is what allows us to identify which bacteria have been taken up in the

plasmid; that is its job.

7. What does the phenotype of the transformed colonies tell you?

It tells us that pGreen is there because it glows under the black light.

8. What one plate would you first inspect to conclude that the transformation occurred successfully? Why?

The plate I would first inspect would be the LB/Amp+plasmid because the selective marker is AMPR and is will grow only in an ampicillin media.

9. Transformation efficiency calculations

a. Determine the total mass of plasmid used.

10 x .005 = .05 ug

b. Calculate the total volume of cell suspension prepared.

.510 ul

c. Now calculate the total volume of total cell suspension that was spread on the

plate.

100/510 = .196

d. Determine the mass of plasmid in the cell suspension spread.

.05 x .196 = .0098

e. Determine the number of colonies per ug plasmid DNA. Express your answer

in scientific notation.

17/.0098 = 1.1735 x 103

10. What factors might influence transformation efficiency? Explain the effect of each

factor you mention.

Transformation efficiency could be affected by the size of the colony added to the solution. In a larger colony the efficiency would increase because there would be more receptive cells. The amount of Luria broth added could also affect efficiency. If the amount of Luria broth was increased, the efficiency would decrease. Another factor would be the amount of AMPR added. The more AMPR added, the higher the efficiency.

Purification of Green Fluorescent Protein Questions

1. What kind of molecule causes the bacteria to turn green?

The kind of molecule that causes the bacteria to turn green is Green Fluorescent

Protein (GFP).

2. Why does transforming the pGREEN plasmid into the bacteria cause the bacteria to turn green?

The bacteria turns green because once the DNA from the plasmid is transformed the Green Fluorescent Protein can be expressed, causing the green glow.

3. What class of molecules does the lysis buffer interact with to release GFP from E.coli

cells?

The class of molecules that the lysis buffer interacts with are the lipids.

4.

a. What aspect of GFP structure allows it to interact so strongly with the HIC

resin?

The aspect of GFP structure that allows it to interact so strongly with the

HIC resin is that part of it is hydrophobic and part is hydrophilic and the salt/ion concentrations.

b. How does the TE buffer release the GFP molecules from the HIC resin in step 15?

The TE buffer releases the GFP from the HIC resin by having chloride ions in the high salt binding buffer repel negative charges in the exterior Beta sheath of the GFP molecule. This repulsion causes the molecule to flip inside out, exposing the hydrophobic chromophore.

5. HIC chromatography does not yield 100 percent pure GFP. What other types of cellular proteins would most likely be found in the GFP preparation?

The other types of cellular proteins most likely found in the GFP preparation would be enzymes because they catalyze all reactions and play a key role in anything involving a protein.

Conclusion:

Overall this lab was effective in allowing a student to perform an actual transformation. As for the potential for error, this lab had several steps that could have tripped a student up. All of the measurements had to be precise and accurate, and the heat shock timing was also a very complicated procedure. Other errors in this lab could have been caused by the concentration of the calcium chloride, the Luria broth, the lysis buffer, and any of the solutions prepared. In conclusion the bacteria treated with the AMPR solution developed a resistance to ampicillin and were able to grow on the LB/Amp+plate. Those that were not treated with the AMPR were not able to grow on this medium. The plates with no ampicillin served as a control to show how the bacteria would look in normal conditions. Transformation is never one hundred percent effective because only cells that are competent enough are able to take up the foreign DNA. That explains why the LB/amp+plasmid plates showed less growth than the control plates.

*all calculations and conclusions about growth are correct... http://sps.k12.ar.us/massengale/examples_of_ap_lab_reports.htm