RFP Lab
Purpose: The purpose of this lab was to show the gene that glows red, Red Fluorescent Protein, that's in jellyfish, in E. Coli. We learned about genetic engineering and got to actually do this experiment ourselves.
Materials & Procedure:
Lab 2A - materials and procedure can be found in Amgen lab manual 2a
Lab 4A - materials and procedure can be found in Amgen lab manual 4a
Lab 5a- materials and procedure can be found in Amgen lab manual part 5a
Lab 6A - materials and procedure can be found in Amgen lab manual 6a
Experimental Overview:
Lab 2A: Verification of Plasmid by Restriction Digest
- We cut a plasmid with BamH1 and HindIII and extracted the RFP-Ara gene.
Lab 4a: Verification of Plasmid Digest by Electrophoresis
- We used electrophoresis to check and see if it was the gene we wanted.
Lab 5a: Transformation of Bacteria with Recombinant Plasmid
- We transformed the bacteria into a recombinant plasmid. We used enzymes to cut and paste the RFP gene.
Lab 6a: Separating the RFP Gene with Column Chromatography
- We used chromatography to separate the RFP from the junk we didn't need.
Lab 2a
1.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 98.6 degrees F because that is human body temperature and enzymes function normally in our body. They are then frozen to stop their growth.
5. In nature, restriction enzymes are found in bacteria to protect them.
6. Bacteria would retain a gene that gives them resistance to antibiotics because then they would live and other bacteria would die. The resistant bacteria require humans to keep editing our antibiotics to keep fighting 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. 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. With this you know which have resistance to kanamycin and ampicillin.
Lab 4a
1. It is useful to use loading dye for this lab to be able to see the plasmid and 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 size so we can guesstimate where they should fall on the ladder.
3. The DNA samples will be visible after the gel has run because the dyes will be on the DNA and made them visible.
4. It is important to check that you have the right recombinant plasmid because using the wrong one in the bacteria would make the wrong thing or not make anything at all.
5. Our gel results were off of the predicted position they would be in.
6. I did see extra bands in the gel, but they could have shown up because we didn't use the proper enzymes or cut the wrong sections.
7. Our gel doesn't show that we used the correct plasmid because we had a lot of extra bands.
8. In the R- lane there is evidence of multiple plasmids because there are multiple bands that are very close to one another.
9. The R+ lane has 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 were which because of the DNA ladder.
11. The lanes with the plasmids seem to have multiple bands bunched together, where as the lanes with linear fragments don't.
Lab 5a
1. The P+ bacteria culture is treated differently because it has more plates than P- so we can compare results. The P- culture is there to see what would happen to bacteria with LB and amp to the bacteria.
2. Cells need time to recover after the heat shock, otherwise they will 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 being contaminated itself.
5. My results matched all of my predictions.
6. There were no red colonies on the LB/amp/ara plate.
7. The red colonies may 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.
Lab 6a
1.) The RFP will be red and will centrifuge to the bottom.
2.) The supernatant is a clear liquid (usually composed of solutions) found on top of a recently microfuged solid. The pellet is this solid and is often made of proteins(RFP).
3.) Proteins are unfolded in the highly salt concentrated buffer. The proteins that don't flow out the column (because they are unfolded and to big now) are refolded when lower salt concentrations are added, thus then they leave the column later.
4.) A protein's conformation is important because it determines the promoter regions of the given protein. These promoter regions are what give each protein their different functions.
5.) The sequence of an amino acid determines how it will fold into a protein.
6.) The RFP-containg elute is brighter than the cell lysate. This is because the elute contains the most RFP, the cell lysate contained all the cell proteins and cell parts along with the RFP.
7.) The red fluorescent protein sticks to the resin column when unfolded (more hydrophobic amino acids).
8.) If we repeated the process with more wash buffers and were more careful about collection, we could increase the red fluorescent protein purity. Also, the size and shape of the column can be modified to maximize good results.
Analysis:
In this lab, we started with the verification of plasmid by restriction digest, then we cut the plasmid with BanH1 and HindIII to cut out RFP-ara from bacterial plasmid. We then, did the verification of plasmid digest by electrophoresis and the transformation of bacteria with recombinant plasmid. At the end of the lab we did the purification of RFP using chromatography.
Reflection:
This lab was really cool to me. This was a huge, long lab that had a lot of different parts to it, some harder than others. We used so many different materials that I have never even heard of before so that was pretty cool. I enjoyed this lab and got to see what it was like to be a real microbiologist. We made some mistakes but overall it was great learning experience.
Materials & Procedure:
Lab 2A - materials and procedure can be found in Amgen lab manual 2a
Lab 4A - materials and procedure can be found in Amgen lab manual 4a
Lab 5a- materials and procedure can be found in Amgen lab manual part 5a
Lab 6A - materials and procedure can be found in Amgen lab manual 6a
Experimental Overview:
Lab 2A: Verification of Plasmid by Restriction Digest
- We cut a plasmid with BamH1 and HindIII and extracted the RFP-Ara gene.
Lab 4a: Verification of Plasmid Digest by Electrophoresis
- We used electrophoresis to check and see if it was the gene we wanted.
Lab 5a: Transformation of Bacteria with Recombinant Plasmid
- We transformed the bacteria into a recombinant plasmid. We used enzymes to cut and paste the RFP gene.
Lab 6a: Separating the RFP Gene with Column Chromatography
- We used chromatography to separate the RFP from the junk we didn't need.
Lab 2a
1.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 98.6 degrees F because that is human body temperature and enzymes function normally in our body. They are then frozen to stop their growth.
5. In nature, restriction enzymes are found in bacteria to protect them.
6. Bacteria would retain a gene that gives them resistance to antibiotics because then they would live and other bacteria would die. The resistant bacteria require humans to keep editing our antibiotics to keep fighting 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. 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. With this you know which have resistance to kanamycin and ampicillin.
Lab 4a
1. It is useful to use loading dye for this lab to be able to see the plasmid and 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 size so we can guesstimate where they should fall on the ladder.
3. The DNA samples will be visible after the gel has run because the dyes will be on the DNA and made them visible.
4. It is important to check that you have the right recombinant plasmid because using the wrong one in the bacteria would make the wrong thing or not make anything at all.
5. Our gel results were off of the predicted position they would be in.
6. I did see extra bands in the gel, but they could have shown up because we didn't use the proper enzymes or cut the wrong sections.
7. Our gel doesn't show that we used the correct plasmid because we had a lot of extra bands.
8. In the R- lane there is evidence of multiple plasmids because there are multiple bands that are very close to one another.
9. The R+ lane has 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 were which because of the DNA ladder.
11. The lanes with the plasmids seem to have multiple bands bunched together, where as the lanes with linear fragments don't.
Lab 5a
1. The P+ bacteria culture is treated differently because it has more plates than P- so we can compare results. The P- culture is there to see what would happen to bacteria with LB and amp to the bacteria.
2. Cells need time to recover after the heat shock, otherwise they will 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 being contaminated itself.
5. My results matched all of my predictions.
6. There were no red colonies on the LB/amp/ara plate.
7. The red colonies may 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.
Lab 6a
1.) The RFP will be red and will centrifuge to the bottom.
2.) The supernatant is a clear liquid (usually composed of solutions) found on top of a recently microfuged solid. The pellet is this solid and is often made of proteins(RFP).
3.) Proteins are unfolded in the highly salt concentrated buffer. The proteins that don't flow out the column (because they are unfolded and to big now) are refolded when lower salt concentrations are added, thus then they leave the column later.
4.) A protein's conformation is important because it determines the promoter regions of the given protein. These promoter regions are what give each protein their different functions.
5.) The sequence of an amino acid determines how it will fold into a protein.
6.) The RFP-containg elute is brighter than the cell lysate. This is because the elute contains the most RFP, the cell lysate contained all the cell proteins and cell parts along with the RFP.
7.) The red fluorescent protein sticks to the resin column when unfolded (more hydrophobic amino acids).
8.) If we repeated the process with more wash buffers and were more careful about collection, we could increase the red fluorescent protein purity. Also, the size and shape of the column can be modified to maximize good results.
Analysis:
In this lab, we started with the verification of plasmid by restriction digest, then we cut the plasmid with BanH1 and HindIII to cut out RFP-ara from bacterial plasmid. We then, did the verification of plasmid digest by electrophoresis and the transformation of bacteria with recombinant plasmid. At the end of the lab we did the purification of RFP using chromatography.
Reflection:
This lab was really cool to me. This was a huge, long lab that had a lot of different parts to it, some harder than others. We used so many different materials that I have never even heard of before so that was pretty cool. I enjoyed this lab and got to see what it was like to be a real microbiologist. We made some mistakes but overall it was great learning experience.