rfp Lab
RFP LABPurpose: To make a red florescent protein from a jellyfish in a bacteria. To also learn about steps in genetic engineering
Materials + Procedure: These can be found in their corresponding areas in the Amgen lab manual.(Ex. 2a materials and procedure are in part 2a for the RFP lab)
EXPERIMENTAL OVERVIEW2a: Preparing the RFP Gene
- In this first lab we separated the RFP-Ara gene from the pAra-R plasmid using BamH1 and HindIII
4a: Electrophoresis to verify plasmid
- Electrophoresis was used to make sure that we had cut the plasmid correctly. We ran the gene and plasmid along with a DNA ladder that will tell you the molecular sizes they should be. We then used the ladder to compare our cut sizes and what they should be.
5a: Transformation of Bacteria using a Recombinant Plasmid
-Here we took restriction enzymes to cut the plasmid and ligase to pastethe RFP gene. We then put on a selective marker to regulate the growth of bacteria that were not resistant to things like Ampicillin. The bacteria were transformed into a recombinant plasmid.
6a: Separating the RFP Gene with Chromatography
- We used a chromatography column to seperate the RFP gene from everything else. There were beads in the column that we used to catch the RFP to seperate it and then extract the gene from the beads with a buffer.
Materials + Procedure: These can be found in their corresponding areas in the Amgen lab manual.(Ex. 2a materials and procedure are in part 2a for the RFP lab)
EXPERIMENTAL OVERVIEW2a: Preparing the RFP Gene
- In this first lab we separated the RFP-Ara gene from the pAra-R plasmid using BamH1 and HindIII
4a: Electrophoresis to verify plasmid
- Electrophoresis was used to make sure that we had cut the plasmid correctly. We ran the gene and plasmid along with a DNA ladder that will tell you the molecular sizes they should be. We then used the ladder to compare our cut sizes and what they should be.
5a: Transformation of Bacteria using a Recombinant Plasmid
-Here we took restriction enzymes to cut the plasmid and ligase to pastethe RFP gene. We then put on a selective marker to regulate the growth of bacteria that were not resistant to things like Ampicillin. The bacteria were transformed into a recombinant plasmid.
6a: Separating the RFP Gene with Chromatography
- We used a chromatography column to seperate the RFP gene from everything else. There were beads in the column that we used to catch the RFP to seperate it and then extract the gene from the beads with a buffer.
Our electrophoresis gel. DNA ladder left, R- middle, R+ right.
RESULTS:
2a: We verified that we had everything we needed for the lab and were prepared for cutting at the end.
Questions:
1. Why does using two different enzymes to cut the plasmid prevent the plasmid from forming a circle without the inserted gene? Without the inserted gene after cutting a plasmid would create an empty spot in the circle and cause the plasmid to never become a full circle.
2. The purpose of setting up a tube without BamH1 HindIII is to create a control for the lab.
3. Enzymes work best at 37 degrees C because that is our body temperature and enzymes are in our body functioning normally. They are then frozen to stunt their growth.
4.
5. In nature, restriction enzymes are found in bacteria to protect them.
6. 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 would retain a gene that gives them resistance to antibiotics because then they would live and other bacteria would die. These resistant bacteria require us to keep changing our antibiotics to keep fighting the bacteria.
7. A gene from humans or a sea anemone can be expressed in bacteria to make a product by cutting out a gene of interest like insulin and cutting out an area for the gene to be inserted into the bacteria. Then the bacteria will make whatever its DNA tells it to.
8. 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. Since the bacteria were mixed together, try to mix the very well so they are both dispersed evenly. Then you can take two portions of the mixture and put each one in a different petri dish. Then you can make a solution to kill of both kinds of bacteria and put one in each. This way some of each kind will live in each dish. Now you know which have resistace to kanamycin and ampicillin.
4a: After running the gels it appeared that the cutting went well and the RFP gene and plasmid were about the proper size based on the DNA ladder.
Questions:
1. It is useful to use loading dye in this lab to be able to see the plasmid and the RFP gene in the gel after running it.
2. We can predict the position of the R- and R+ tubes based on the DNA ladder because we do know their weights so we can gauge where they should fall on the ladder.
3. The DNA samples will be visible after the gel has run because the dyes will have dispersed onto the DNA and have made them visible to us.
4. It is important to verify that you have the correct recombinant plasmid because using the wrong one in the bacteria would produce the wrong product or not produce anything at all.
5. Our gel results were a little off of the predicted position they should be in, but it was close.
6. I did not see any extra bands in the gel, but they could show up by not using the proper enzymes and then they cut sections we did not want cut.
7. Our gel does show that we used the correct plasmid because it is where it is supposed to be based on the DNA ladder.
8. In the R- lane there is evidence of maybe multiple plasmids because there are multiple bands that are very close to one another, but show separation.
9. The R+ lane appears to have complete digestion because we can see the RFP gene away from the plasmid.
10. We would expect to find the rfp gene and ampR in the R+ lane. We could see each of them and knew which ones were which based on the DNA ladder and knowledge of their sizes.
11. The lanes with the plasmids appear to have multiple bands clumped together, where as the lanes with linear fragments do not.
5a: The bacteria appeared to have accepted the changes made to it and the correct bacteria lived based on the conditions that the bacteria was in.
Questions:
1. The P+ bacteria culture is treated differently because it has more plates than P- for the sake of comparing results. The P- culture is there to see the effects of LB and amp to the bacteria.
2. Cells need time to recover after the heat shock, otherwise they might die if they can't re-stabilize.
3. Cells are incubated at 37 degrees C because that is a normal temperature condition.
4. Aseptic is important in this lab because it keeps the bacteria from contaminating and becoming contaminated itself.
5. Most of my results except for the P+ only plate matched my predictions. I realized that the P+ was resistant to ara and amp, unlike I thought they were.
6. There were no red colonies on the LB/amp/ara plate.
7. The red colonies might only appear on the LB/amp/ara plate and not the LB/amp plate because it needs ara to survive and make the rfp.
8. It is important to have many copies of a recombinant plasmid in a cell to create more protein in a cell.
9. The rfp gene makes specialized proteins that develop into the traits of an organism.
10. Bacteria can make any protein because they are made to produce proteins, and if they are given the right codes, they will make any proteins.
6a: The RFP easily binded to the beads to separate it and then we were able to obtain the RFP gene by itself with ease.
Questions:
1. You can determine where the rfp is in each step by watching for the red substance as it travels.
2. The supernatant is a bunch of clear cell nutrients and the pellet is compact bacterial cells with rfp.
3. The BB binds RFP to the resin bed, the WB washes away unwanted proteins, and EB knocks off RFP at the end.
4. A protein's conformation is important to it functioning because it needs to conform with its surroundings, otherwise it won't fit in and function with everything else like it should be.
5. In proteins, amino acids decide how the protein will fold based on how amino acids bond and their polarity.
6. There was not a noticeable difference before and after putting the rfp in the column as far as its color
7. The beads in the column could be made so they will bind very well with the rfp and have more of them to ensure that they all get on and increase the purity of the rfp sample.
RESULTS:
2a: We verified that we had everything we needed for the lab and were prepared for cutting at the end.
Questions:
1. Why does using two different enzymes to cut the plasmid prevent the plasmid from forming a circle without the inserted gene? Without the inserted gene after cutting a plasmid would create an empty spot in the circle and cause the plasmid to never become a full circle.
2. The purpose of setting up a tube without BamH1 HindIII is to create a control for the lab.
3. Enzymes work best at 37 degrees C because that is our body temperature and enzymes are in our body functioning normally. They are then frozen to stunt their growth.
4.
5. In nature, restriction enzymes are found in bacteria to protect them.
6. 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 would retain a gene that gives them resistance to antibiotics because then they would live and other bacteria would die. These resistant bacteria require us to keep changing our antibiotics to keep fighting the bacteria.
7. A gene from humans or a sea anemone can be expressed in bacteria to make a product by cutting out a gene of interest like insulin and cutting out an area for the gene to be inserted into the bacteria. Then the bacteria will make whatever its DNA tells it to.
8. 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. Since the bacteria were mixed together, try to mix the very well so they are both dispersed evenly. Then you can take two portions of the mixture and put each one in a different petri dish. Then you can make a solution to kill of both kinds of bacteria and put one in each. This way some of each kind will live in each dish. Now you know which have resistace to kanamycin and ampicillin.
4a: After running the gels it appeared that the cutting went well and the RFP gene and plasmid were about the proper size based on the DNA ladder.
Questions:
1. It is useful to use loading dye in this lab to be able to see the plasmid and the RFP gene in the gel after running it.
2. We can predict the position of the R- and R+ tubes based on the DNA ladder because we do know their weights so we can gauge where they should fall on the ladder.
3. The DNA samples will be visible after the gel has run because the dyes will have dispersed onto the DNA and have made them visible to us.
4. It is important to verify that you have the correct recombinant plasmid because using the wrong one in the bacteria would produce the wrong product or not produce anything at all.
5. Our gel results were a little off of the predicted position they should be in, but it was close.
6. I did not see any extra bands in the gel, but they could show up by not using the proper enzymes and then they cut sections we did not want cut.
7. Our gel does show that we used the correct plasmid because it is where it is supposed to be based on the DNA ladder.
8. In the R- lane there is evidence of maybe multiple plasmids because there are multiple bands that are very close to one another, but show separation.
9. The R+ lane appears to have complete digestion because we can see the RFP gene away from the plasmid.
10. We would expect to find the rfp gene and ampR in the R+ lane. We could see each of them and knew which ones were which based on the DNA ladder and knowledge of their sizes.
11. The lanes with the plasmids appear to have multiple bands clumped together, where as the lanes with linear fragments do not.
5a: The bacteria appeared to have accepted the changes made to it and the correct bacteria lived based on the conditions that the bacteria was in.
Questions:
1. The P+ bacteria culture is treated differently because it has more plates than P- for the sake of comparing results. The P- culture is there to see the effects of LB and amp to the bacteria.
2. Cells need time to recover after the heat shock, otherwise they might die if they can't re-stabilize.
3. Cells are incubated at 37 degrees C because that is a normal temperature condition.
4. Aseptic is important in this lab because it keeps the bacteria from contaminating and becoming contaminated itself.
5. Most of my results except for the P+ only plate matched my predictions. I realized that the P+ was resistant to ara and amp, unlike I thought they were.
6. There were no red colonies on the LB/amp/ara plate.
7. The red colonies might only appear on the LB/amp/ara plate and not the LB/amp plate because it needs ara to survive and make the rfp.
8. It is important to have many copies of a recombinant plasmid in a cell to create more protein in a cell.
9. The rfp gene makes specialized proteins that develop into the traits of an organism.
10. Bacteria can make any protein because they are made to produce proteins, and if they are given the right codes, they will make any proteins.
6a: The RFP easily binded to the beads to separate it and then we were able to obtain the RFP gene by itself with ease.
Questions:
1. You can determine where the rfp is in each step by watching for the red substance as it travels.
2. The supernatant is a bunch of clear cell nutrients and the pellet is compact bacterial cells with rfp.
3. The BB binds RFP to the resin bed, the WB washes away unwanted proteins, and EB knocks off RFP at the end.
4. A protein's conformation is important to it functioning because it needs to conform with its surroundings, otherwise it won't fit in and function with everything else like it should be.
5. In proteins, amino acids decide how the protein will fold based on how amino acids bond and their polarity.
6. There was not a noticeable difference before and after putting the rfp in the column as far as its color
7. The beads in the column could be made so they will bind very well with the rfp and have more of them to ensure that they all get on and increase the purity of the rfp sample.
Gel Sample to see RFP purity and correctness of size. We were the furthest to the right lane.
DATA/GEL ANALYSIS:
Everything went very well in the lab. In the end we were able to insert the rfp gene into bacteria and have them produce rfp. Then we were able to get a good sample of it in. We had some issues with the products given to us, but it worked out in the end. The gel sample to check the RFP purity was less pure than it could have been. There are several bands in our lane, which shows that there were proteins other than the RFP that got in. The RFP might not have been as pure as some of the other ones because we took the RFP out of the columns too early, or not enough times to remove all of the other substances and isolate the RFP. The size, however, of the thickest band(RFP) was around the correct size that it should have been.
REFLECTION:
Our group worked very well together and we got everything done well and made plans. We had problems trying to answer the questions in the lab together though. The actual labs went smoothly and we got good results in the end, even if they were a little off target. This lab was enjoyable and interesting, but would have been better with background knowledge or just more information as far as what was happening and how.
DATA/GEL ANALYSIS:
Everything went very well in the lab. In the end we were able to insert the rfp gene into bacteria and have them produce rfp. Then we were able to get a good sample of it in. We had some issues with the products given to us, but it worked out in the end. The gel sample to check the RFP purity was less pure than it could have been. There are several bands in our lane, which shows that there were proteins other than the RFP that got in. The RFP might not have been as pure as some of the other ones because we took the RFP out of the columns too early, or not enough times to remove all of the other substances and isolate the RFP. The size, however, of the thickest band(RFP) was around the correct size that it should have been.
REFLECTION:
Our group worked very well together and we got everything done well and made plans. We had problems trying to answer the questions in the lab together though. The actual labs went smoothly and we got good results in the end, even if they were a little off target. This lab was enjoyable and interesting, but would have been better with background knowledge or just more information as far as what was happening and how.