Red Fluorescent Protein Lab
Purpose
To express red fluorescent protein from jellyfish in bacteria. This expirement would also help us learn about genetic engineering.
To express red fluorescent protein from jellyfish in bacteria. This expirement would also help us learn about genetic engineering.
Materials
Lab 2a
Materials can be found in Amgen lab manual part 2a. |
Lab 4a
Materials can be found in Amgen lab manual part 4a. |
Lab 5a
Materials can be found in Amgen lab manual part 5a. |
Lab 6
Materials can be found in Amgen lab manual part 6. |
Experimental Overview
Lab 2a - We verified that we had the correct plasma by using a restriction digest. We cut the plasmid with BamHI and Hind III.
Lab 4a - We verified the plasmid digest by electrophoresis.
Lab 5a - Then we transformed the bacteria with a recombinant plasmid.
Lab 6 - The we purified the RFP using chromotography.
Lab 2a - We verified that we had the correct plasma by using a restriction digest. We cut the plasmid with BamHI and Hind III.
Lab 4a - We verified the plasmid digest by electrophoresis.
Lab 5a - Then we transformed the bacteria with a recombinant plasmid.
Lab 6 - The we purified the RFP using chromotography.
Data Results
Before the 2a Lab:
1. If pARA-R is digested with BamHI and HindIII, what fragments are produced? Record the nucleotide sequence of the sticky ends and the length of each fragment (bp), and indicate the genes and other important sequences present on each fragment. There are two fragments are produced. They are RFP with pBAD and Ara-C with ori with Amp-R. The RFP plus pBAD is 807 BP, and Ara-C, ori, and Amp-R is 4495 BP.
2. In order to create a plasmid that can produce the red fluorescent protein in
bacteria, what components are needed in the plasmid? We need the RFP gene and Ara-C.
3. If the uptake of DNA by bacteria is inefficient (as discussed in the reading), why is a selectable marker (gene with resistance to an antibiotic) critical in cloning a gene in bacteria? The selectable marker allows only the desired bacteria to grow. It separates the bacteria from the desired gene.
2a Questions:
1. List in words or indicate in a drawing the important features of a plasmid vector that are required to clone a gene. Explain the purpose of each feature. Ori = origin of replication; RFP = red fluorescent protein (gene of interest); Amp-R = selectable marker; Ara-C = binds to promoter so we get transcription of gene of interest.
2. What role do restriction enzymes have in nature? Restriction enzymes are a defense mechanism. They cut up foreign bodies.
3. Using your understanding of evolution, why would bacteria retain a gene that gives them resistance to antibiotics? How is the existence of bacteria with antibiotic resistance affecting medicine today?
Bacteria retain genes that give them resistance to antibiotics to protect themselves from disease.
4. Bacteria, sea anemones, and humans seem, on the surface, to be very different organisms. Explain how a gene from humans or a sea anemone can be expressed in bacteria to make a product never before made in bacteria. The central dogma is the same in all organisms.
5. Due to a mishap in the lab, bacteria carrying a plasmid with an ampicillin resistant gene and bacteria carrying a plasmid with a gene that provides resistance to another antibiotic (kanamycin) were accidentally mixed together. Design an experiment that will allow you to sort out the two kinds of bacteria. Create a petry dish that grows both Kan and Amp bacteria. Put half of the mixed bacteria into each petry dish and wait a day. The bacteria should have died and then you should be able to separate them apart.
4a Before the Lab Questions:
1. The pARA-R plasmid you digested in Laboratory 2A was replicated in a bacterial cell. What configurations—supercoiled, nicked circle, and multimer—might the plasmid have before digestion?
The plasma could have been any of these configurations.
2. You need to catalog all the products you might see, including the different plasmid configurations. Review your work in Laboratory 2A. What products might you expect to see in the R– and R+ tubes? Create a table that shows all the possible fragments and plasmids by tube. Include the length (bp size) of each possible fragment or plasmid, and arrange the products found in each microfuge tube by size, from smallest to largest. Include any possible plasmid configurations, and arrange them first by size and next by speed through the gel, from fastest to slowest.
R+ Tube: pARA-R is 302 BP. It can be any combination. The plasma is supercoiled and it is the fastest through the gel.
R- Tube: RFP and -pBAD is 807 BP. There is also AMP-R, Ara-C, and Ori is 4495 BP.
4a Questions:
1. Why is it important to verify that you have the correct recombinant plasmid?
It is important to verify that you have the correct recombinant plasmid in case you did something wrong in the procedure.
2. How did your actual gel results compare to your gel predictions?
1. If pARA-R is digested with BamHI and HindIII, what fragments are produced? Record the nucleotide sequence of the sticky ends and the length of each fragment (bp), and indicate the genes and other important sequences present on each fragment. There are two fragments are produced. They are RFP with pBAD and Ara-C with ori with Amp-R. The RFP plus pBAD is 807 BP, and Ara-C, ori, and Amp-R is 4495 BP.
2. In order to create a plasmid that can produce the red fluorescent protein in
bacteria, what components are needed in the plasmid? We need the RFP gene and Ara-C.
3. If the uptake of DNA by bacteria is inefficient (as discussed in the reading), why is a selectable marker (gene with resistance to an antibiotic) critical in cloning a gene in bacteria? The selectable marker allows only the desired bacteria to grow. It separates the bacteria from the desired gene.
2a Questions:
1. List in words or indicate in a drawing the important features of a plasmid vector that are required to clone a gene. Explain the purpose of each feature. Ori = origin of replication; RFP = red fluorescent protein (gene of interest); Amp-R = selectable marker; Ara-C = binds to promoter so we get transcription of gene of interest.
2. What role do restriction enzymes have in nature? Restriction enzymes are a defense mechanism. They cut up foreign bodies.
3. Using your understanding of evolution, why would bacteria retain a gene that gives them resistance to antibiotics? How is the existence of bacteria with antibiotic resistance affecting medicine today?
Bacteria retain genes that give them resistance to antibiotics to protect themselves from disease.
4. Bacteria, sea anemones, and humans seem, on the surface, to be very different organisms. Explain how a gene from humans or a sea anemone can be expressed in bacteria to make a product never before made in bacteria. The central dogma is the same in all organisms.
5. Due to a mishap in the lab, bacteria carrying a plasmid with an ampicillin resistant gene and bacteria carrying a plasmid with a gene that provides resistance to another antibiotic (kanamycin) were accidentally mixed together. Design an experiment that will allow you to sort out the two kinds of bacteria. Create a petry dish that grows both Kan and Amp bacteria. Put half of the mixed bacteria into each petry dish and wait a day. The bacteria should have died and then you should be able to separate them apart.
4a Before the Lab Questions:
1. The pARA-R plasmid you digested in Laboratory 2A was replicated in a bacterial cell. What configurations—supercoiled, nicked circle, and multimer—might the plasmid have before digestion?
The plasma could have been any of these configurations.
2. You need to catalog all the products you might see, including the different plasmid configurations. Review your work in Laboratory 2A. What products might you expect to see in the R– and R+ tubes? Create a table that shows all the possible fragments and plasmids by tube. Include the length (bp size) of each possible fragment or plasmid, and arrange the products found in each microfuge tube by size, from smallest to largest. Include any possible plasmid configurations, and arrange them first by size and next by speed through the gel, from fastest to slowest.
R+ Tube: pARA-R is 302 BP. It can be any combination. The plasma is supercoiled and it is the fastest through the gel.
R- Tube: RFP and -pBAD is 807 BP. There is also AMP-R, Ara-C, and Ori is 4495 BP.
4a Questions:
1. Why is it important to verify that you have the correct recombinant plasmid?
It is important to verify that you have the correct recombinant plasmid in case you did something wrong in the procedure.
2. How did your actual gel results compare to your gel predictions?
Our gel did not match our predictions at all. It was very difficult to see because the protein was transparent and the visible protein we saw was in an odd line instead of being in blocks. The loading dye did not show up, the R- was faint, and the R+ was visible.
3. Do you see any bands that are not expected? What could explain the origin of these unexpected bands?
We only had the missing loading dye.
4. Does the gel photograph show that you are using the correct recombinant plasmid? Describe the evidence you used to make this assessment.
Our photo does not show that we are using the correct recombinant plasmid because the line of protein is not at the right spot.
5. In the R- lane, do you see evidence of multiple configurations of plasmids? Explain your answer.
We saw evidence of multiple configurations because there were two different fragments of the R- plasmid.
6. In the R+ lane, do you see evidence of complete digestion? Explain your answer.
We could not discern whether or not there was evident of complete digestion because the fragment had turned into a line.
7. Where would you expect to see the RFP gene and the Amp-R gene in the gel photograph? Are you able to locate these two genes? Explain your answer.
We should have seen a band in the R+ lane around 807 BP as well as a band at around 4,495 BP. We were unable to locate these two genes because our band had turned into a line stretching from about 350 BP to 5500 BP.
8. Compare the lanes that have linear fragments with the lanes that have plasmids. Is there a difference in the shape of the bands between these two DNA forms?
We were unable to tell if the plasmids had different shape due to the dis-formation of the bands.
We only had the missing loading dye.
4. Does the gel photograph show that you are using the correct recombinant plasmid? Describe the evidence you used to make this assessment.
Our photo does not show that we are using the correct recombinant plasmid because the line of protein is not at the right spot.
5. In the R- lane, do you see evidence of multiple configurations of plasmids? Explain your answer.
We saw evidence of multiple configurations because there were two different fragments of the R- plasmid.
6. In the R+ lane, do you see evidence of complete digestion? Explain your answer.
We could not discern whether or not there was evident of complete digestion because the fragment had turned into a line.
7. Where would you expect to see the RFP gene and the Amp-R gene in the gel photograph? Are you able to locate these two genes? Explain your answer.
We should have seen a band in the R+ lane around 807 BP as well as a band at around 4,495 BP. We were unable to locate these two genes because our band had turned into a line stretching from about 350 BP to 5500 BP.
8. Compare the lanes that have linear fragments with the lanes that have plasmids. Is there a difference in the shape of the bands between these two DNA forms?
We were unable to tell if the plasmids had different shape due to the dis-formation of the bands.
5a Before the Lab Questions:
1. Ampicillin is an antibiotic that kills bacterial cells by disrupting the formation of cell walls. However, the pARA-R plasmid has the ampicillin resistance gene, which produces a protein that breaks down ampicillin. What is the purpose of growing bacteria that have been transformed in the presence of ampicillin?
The purpose of growing bacteria that resists ampicillin is to make sure that the desired cell lives.
2. What will happen when bacterial cells that contain the pARA-R plasmid are not given arabinose?
If the bacterial cells are not given arabinose, then the pARA-R plasmid will not turn on the promoter. Then the protein will not turn red.
3. In the lab, you will add samples of the control group P– and the treatment group P+ to plates that contain various combinations of Luria Broth (LB), ampicillin, and the sugar arabinose. The plates will be arranged as follows:
In the LB plate, both the P- and P+ will grow. In the LB/Amp plate, no P- will grow, but P+ will. In the LB/ Amp/Ara plate, there will barely any bacteria growth.
5a Before the Lab:
1. Ampicillin is an antibiotic that kills bacterial cells by disrupting the formation of cell walls. However, the pARA-R plasmid has the ampicillin resistance gene, which produces a protein that breaks down ampicillin. What is the purpose of growing bacteria that have been transformed in the presence of ampicillin?
The purpose of growing these bacteria is to separate out which have ampicillin and which don't.
2. What will happen when bacterial cells that contain the pARA-R plasmid are not given arabinose?
If the bacteria cells are not given arabinose, then the RFP gene will not be expressed.
3. In the lab, you will add samples of the control group P– and the treatment group P+ to plates that contain various combinations of Luria Broth (LB), ampicillin, and the sugar arabinose. Predict the growth on each dish.
I predict that Plate I (LB) will have non-glowing growth on both sides. I predict that Plate II (LB/Amp) will have non-glowing growth on the P+ side. For Plate III (LB/Amp/Ara), I predict a glowing red colony.
5a Questions:
1.In the lab, you will add samples of the control group P– and the treatment group P+ to plates that contain various combinations of Luria Broth (LB), ampicillin, and the sugar arabinose. The plates will be arranged as follows:
Our predictions sort of matched our results. The LB plate had limited growth. The LB/Amp and LB/Amp/Ara plate matched our prediction.
2. How many red colonies were present on your LB/amp/ara plate?
There were no red colonies visible either due to temperature or not enough time in the incubator.
3. Why did the red colonies only appear on the LB/amp/ara plate and not the
LB/amp plate?
The red colonies would have only appeared on the plate with araganose because araganose allows the red protein to be expressed.
4. 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?
The more copies there are, then the chance is better that the gene of interest will be expressed.
5. How is the information encoded in the RFP gene expressed as a trait? Be sure to use what you have previously learned about gene expression and the relationship between DNA, RNA, protein, and traits.
The RFP gene is expressed as a trait through transcription (central dogma).
6. Why is it possible for bacteria to make a human protein, such as insulin, or a sea anemone protein, such as the red fluorescent dye?
Central Dogma.
6a Before the Lab:
1. How can solutions of different salt concentrations, which will unfold proteins to varying degrees, be used to help purify red fluorescent protein using column chromatography?
The folded protein will be diffused out of the column, taking the RFP gene with it.
6a Questions:
1. Why is a protein’s conformation important for carrying out its function?
A protein's conformation tells you what its promoter is.
2. What properties of the amino acids in a protein relate to protein folding?
Amino acids tell how the protein will fold.
3. Does the eluate containing your red fluorescent protein appear less bright or brighter than it did in the cell lysate following centrifugation? If there is a noticeable difference in the intensity of the red color, what might account for that?
The RFP eluate is bighter than it was in the cell lysate because we diffused the unwanted protein out.
4. What characteristic of red fluorescent protein is used as the basis for separation by column chromatography?
When unfolded the RFP stick to the resin column.
5. How might the column chromatography procedure be adjusted or modified to increase the purity of the red fluorescent protein sample?
To increase the purity of the RFP, we could continue to wash it in column chromotography.
6b Questions:
1. Binding Buffer (BB): causes amino acid and protein bind to the resin beads.
Wash Buffer (WB): removes loose proteins that are not bound to the resin beads.
Elution Buffer (EB): takes off protein off resin beads.
Column Equilibration Buffer (CEB): stores resin beads.
2. This time, the supernatant was more pink than clear. The pellet was a little darker pink than the supernatant.
1. Ampicillin is an antibiotic that kills bacterial cells by disrupting the formation of cell walls. However, the pARA-R plasmid has the ampicillin resistance gene, which produces a protein that breaks down ampicillin. What is the purpose of growing bacteria that have been transformed in the presence of ampicillin?
The purpose of growing bacteria that resists ampicillin is to make sure that the desired cell lives.
2. What will happen when bacterial cells that contain the pARA-R plasmid are not given arabinose?
If the bacterial cells are not given arabinose, then the pARA-R plasmid will not turn on the promoter. Then the protein will not turn red.
3. In the lab, you will add samples of the control group P– and the treatment group P+ to plates that contain various combinations of Luria Broth (LB), ampicillin, and the sugar arabinose. The plates will be arranged as follows:
In the LB plate, both the P- and P+ will grow. In the LB/Amp plate, no P- will grow, but P+ will. In the LB/ Amp/Ara plate, there will barely any bacteria growth.
5a Before the Lab:
1. Ampicillin is an antibiotic that kills bacterial cells by disrupting the formation of cell walls. However, the pARA-R plasmid has the ampicillin resistance gene, which produces a protein that breaks down ampicillin. What is the purpose of growing bacteria that have been transformed in the presence of ampicillin?
The purpose of growing these bacteria is to separate out which have ampicillin and which don't.
2. What will happen when bacterial cells that contain the pARA-R plasmid are not given arabinose?
If the bacteria cells are not given arabinose, then the RFP gene will not be expressed.
3. In the lab, you will add samples of the control group P– and the treatment group P+ to plates that contain various combinations of Luria Broth (LB), ampicillin, and the sugar arabinose. Predict the growth on each dish.
I predict that Plate I (LB) will have non-glowing growth on both sides. I predict that Plate II (LB/Amp) will have non-glowing growth on the P+ side. For Plate III (LB/Amp/Ara), I predict a glowing red colony.
5a Questions:
1.In the lab, you will add samples of the control group P– and the treatment group P+ to plates that contain various combinations of Luria Broth (LB), ampicillin, and the sugar arabinose. The plates will be arranged as follows:
Our predictions sort of matched our results. The LB plate had limited growth. The LB/Amp and LB/Amp/Ara plate matched our prediction.
2. How many red colonies were present on your LB/amp/ara plate?
There were no red colonies visible either due to temperature or not enough time in the incubator.
3. Why did the red colonies only appear on the LB/amp/ara plate and not the
LB/amp plate?
The red colonies would have only appeared on the plate with araganose because araganose allows the red protein to be expressed.
4. 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?
The more copies there are, then the chance is better that the gene of interest will be expressed.
5. How is the information encoded in the RFP gene expressed as a trait? Be sure to use what you have previously learned about gene expression and the relationship between DNA, RNA, protein, and traits.
The RFP gene is expressed as a trait through transcription (central dogma).
6. Why is it possible for bacteria to make a human protein, such as insulin, or a sea anemone protein, such as the red fluorescent dye?
Central Dogma.
6a Before the Lab:
1. How can solutions of different salt concentrations, which will unfold proteins to varying degrees, be used to help purify red fluorescent protein using column chromatography?
The folded protein will be diffused out of the column, taking the RFP gene with it.
6a Questions:
1. Why is a protein’s conformation important for carrying out its function?
A protein's conformation tells you what its promoter is.
2. What properties of the amino acids in a protein relate to protein folding?
Amino acids tell how the protein will fold.
3. Does the eluate containing your red fluorescent protein appear less bright or brighter than it did in the cell lysate following centrifugation? If there is a noticeable difference in the intensity of the red color, what might account for that?
The RFP eluate is bighter than it was in the cell lysate because we diffused the unwanted protein out.
4. What characteristic of red fluorescent protein is used as the basis for separation by column chromatography?
When unfolded the RFP stick to the resin column.
5. How might the column chromatography procedure be adjusted or modified to increase the purity of the red fluorescent protein sample?
To increase the purity of the RFP, we could continue to wash it in column chromotography.
6b Questions:
1. Binding Buffer (BB): causes amino acid and protein bind to the resin beads.
Wash Buffer (WB): removes loose proteins that are not bound to the resin beads.
Elution Buffer (EB): takes off protein off resin beads.
Column Equilibration Buffer (CEB): stores resin beads.
2. This time, the supernatant was more pink than clear. The pellet was a little darker pink than the supernatant.
Data Analysis
After purifying the gels, we started to examine our them. We were shocked to see that our bacteria was not red, but in fact almost transparent. While the bacteria was not glowing, we still managed to grow a colony. This was accomplished by using restriction enzymes to cut out the RFP gene and confirming that we had had the right gene though gel electrophoresis.
This chart is a protein ladder. It shows how much protein is in the gel
This here is a gel of a group from 2&3 period. Our gel came out very faint, so we thought it would be best to show an better example of what a good gel should look like. In this gel, it is easy to see the bands of extracted protein. If you compare the chart with the gel, you would see that it
Reflection
1. What did you like/find interesting?
I found it interesting that you can decide what bacteria can grow on a plate. I have heard of it before but to actually do this made it that much better. I found it interesting how the plates grew the bacteria. I am curious how the plates can grow the bacteria.
2. How did you and your partner collaborate?
My group worked well together. We were almost always on task, and if one of us had any questions about the expiriement, one of us would always know what was going on and answer the question. This was one of the better groups I've had for a lab so far.
3. What would you do differently next time?
Next time, I would to have liked to use the proper amounts of BB, WB, EB, and CEB. It seemed like we never had enough of it, so our pellet seemed like it was more clear than pink.
This lab was very long. The multiple steps to getting the RFP was sometimes very confusing. I believe that this was the toughest lab we've ever done because of its complexity and length. I did find it interesting that all of these steps were necessary in acquiring the gene of interest. There was no easy way to get it. All in all, this lab was fun and definitely a brain teaser. I enjoyed the task of this lab. I just wish our final gel came out better.
I found it interesting that you can decide what bacteria can grow on a plate. I have heard of it before but to actually do this made it that much better. I found it interesting how the plates grew the bacteria. I am curious how the plates can grow the bacteria.
2. How did you and your partner collaborate?
My group worked well together. We were almost always on task, and if one of us had any questions about the expiriement, one of us would always know what was going on and answer the question. This was one of the better groups I've had for a lab so far.
3. What would you do differently next time?
Next time, I would to have liked to use the proper amounts of BB, WB, EB, and CEB. It seemed like we never had enough of it, so our pellet seemed like it was more clear than pink.
This lab was very long. The multiple steps to getting the RFP was sometimes very confusing. I believe that this was the toughest lab we've ever done because of its complexity and length. I did find it interesting that all of these steps were necessary in acquiring the gene of interest. There was no easy way to get it. All in all, this lab was fun and definitely a brain teaser. I enjoyed the task of this lab. I just wish our final gel came out better.