RFP (Red Fluorescent Protein) Lab
Purpose: Make RFP from jellyfish in bacteria and learn about the steps of genetic engineering
Materials & procedure (link for lab manual below):
Look at labs 2a, 4a, 5a, 6a
Materials & procedure (link for lab manual below):
Look at labs 2a, 4a, 5a, 6a
Experimental Overview:
-Part 2a: To ensure that we have the correct recombinant plasmid we have been given is the correct one to make the red fluorescent protein.
-Part 4a: To use gel electrophoresis to look at the products from the restriction digest of the pARA-R plasmid.
-Part 5a: To transform the E-coli bacteria with the pARA-R plasmid.
-Part 6a: To break open the cells and purify the protein.
Results:
Lab 2a:
Pre-lab Questions:
Post-lab Questions:
1. Plasmid Vector
3. They would keep the gene because it protects them from the antibiotic so that it doesn't kill them. It is affecting medicine because we need stronger and stronger antibodies to kill them.
4. All genes have a central dogma (DNA- mRNA- Protein), so they can be expressed in different organisms.
5. Put half a colony in an ampicillin petri dish and the other in a kanamycin petri dish. The ampicillin dish will be left with ampicillin- resistant bacteria and the other dish will only have kanamycin- resistant bacteria.
Lab 4a:
Pre- Lab Questions:
-Part 2a: To ensure that we have the correct recombinant plasmid we have been given is the correct one to make the red fluorescent protein.
-Part 4a: To use gel electrophoresis to look at the products from the restriction digest of the pARA-R plasmid.
-Part 5a: To transform the E-coli bacteria with the pARA-R plasmid.
-Part 6a: To break open the cells and purify the protein.
Results:
Lab 2a:
Pre-lab Questions:
- The fragments that are produced are the RFP pBAD and Ara-C fragment with ori and Amp-R. The nucleotide sequence length of the RFP fragment is 807 BP and the Ara-C fragment is 4495 BP.
- The RFP gene and the Ara-C (which binds to the promoter gene) are needed.
- It is important because it determines which bacteria will continue to grow and which dies to separate the colonies.
Post-lab Questions:
1. Plasmid Vector
- Ori= Origin of replication
- RFP and PBad= Genes of interest
- Amp-R= Selective Marker
- Ara-C= Binds to the promoter region which leads to transcription
3. They would keep the gene because it protects them from the antibiotic so that it doesn't kill them. It is affecting medicine because we need stronger and stronger antibodies to kill them.
4. All genes have a central dogma (DNA- mRNA- Protein), so they can be expressed in different organisms.
5. Put half a colony in an ampicillin petri dish and the other in a kanamycin petri dish. The ampicillin dish will be left with ampicillin- resistant bacteria and the other dish will only have kanamycin- resistant bacteria.
Lab 4a:
Pre- Lab Questions:
- The plasmid can have all three configurations: supercoiled, nicked circle, and multimer.
2.
Tube R+ |
Fragments (in order from increasing BP size) pARA-R: 5, 302 BP Can have all three combinations supercoiled = fastest |
R-
|
1. RFP- pBAD: 807 BP
2. Amp-R, Ori, Ara-C: 4,495 BP |
3.
Flowchart on how to do this Lab
Flowchart on how to do this Lab
Post- Lab Questions:
1. It is important to verify that we have the right recombinant plasmid because we could make measuring mistakes, our materials could be faulty, or make mistakes during the procedure.
2. Our R+ and R- columns are perfect, but our loading dye column did not show at all.
1. It is important to verify that we have the right recombinant plasmid because we could make measuring mistakes, our materials could be faulty, or make mistakes during the procedure.
2. Our R+ and R- columns are perfect, but our loading dye column did not show at all.
3. We don't have any unexpected bands but we have an unexpected blank space.
4. The above photograph shows that we are using the correct recombinant plasmid because the R+ has two correctly placed fragments.
5. Two different bants prove multiple plasmid configurations.
6. It was completely digested because there are only two fragments in the R+ lane.
7. In the R+ lane, we expect to see a band around 807 bp as well as a band at around 4,495 bp. We were able to locate these two genes as evident in our picture.
8. The bands for the linear fragments have trailing edges instead of the non-trailing edges of the lanes that have plasmids.
Lab 5a
Pre-Lab Questions:
1. They do that because they need to separate the the two kinds of genes.
2. When they don't have arabinose the gene won't be expressed, so there won't be a red glow.
3. I predict that the LB plate will have non-glowing growth on both sides. I predict that the LB/Amp plate will have non-glowing growth on the P+ side. For the LB/Amp/Ara plate, I predict a glowing red colony.
Post-Lab Questions:
1. Most of our results matched my prediction but the LB/Amp/Ara plate wasn't glowing red like it was supposed to. This happened because the company shipped us bad materials so this happened to everyone, not just us.
4. The above photograph shows that we are using the correct recombinant plasmid because the R+ has two correctly placed fragments.
5. Two different bants prove multiple plasmid configurations.
6. It was completely digested because there are only two fragments in the R+ lane.
7. In the R+ lane, we expect to see a band around 807 bp as well as a band at around 4,495 bp. We were able to locate these two genes as evident in our picture.
8. The bands for the linear fragments have trailing edges instead of the non-trailing edges of the lanes that have plasmids.
Lab 5a
Pre-Lab Questions:
1. They do that because they need to separate the the two kinds of genes.
2. When they don't have arabinose the gene won't be expressed, so there won't be a red glow.
3. I predict that the LB plate will have non-glowing growth on both sides. I predict that the LB/Amp plate will have non-glowing growth on the P+ side. For the LB/Amp/Ara plate, I predict a glowing red colony.
Post-Lab Questions:
1. Most of our results matched my prediction but the LB/Amp/Ara plate wasn't glowing red like it was supposed to. This happened because the company shipped us bad materials so this happened to everyone, not just us.
2. There were no red colonies on the plate.
3. The red colonies would have only appeared on the araganose because it allows the red protein to be expressed.
4.The more copies there are, the more products there are.
5. The central dogma (DNA- RNA- Protein) ends with a protein. The RFP protein is expressed in the form of the red glow as a trait.
6. All genes have the same central dogma.
Lab 6a:
Pre-Lab Question:
1. Proteins are unfolded in the highly salt concentrated buffer. The proteins that don't flow out the column are refolded when lower salt concentrations are added, so they leave the column.
Post-Lab Questions:
1. It determines the promoter regions of the given protein. These promoter regions are what give each protein their different functions.
2. The sequence determines how it will fold in a protein
3. The RFP-containing elute is brighter than the cell lysate because the elute contains the most RFP while the cell lysate contained all the cell proteins.
4. The red fluorescent protein sticks to the resin column when unfolded
5. To increase the purity of the red fluorescent protein sample, we could use more wash buffers and be more careful about collection.
Analysis/ Conclusion:
In this lab, we tried to make a red fluorescent bacteria colony. But we could not make it glow red because we got faulty material so it wasn't any of our fault. We still were able to make the protein but not use it for what it was supposed to. So in terms of actually getting it to work, we failed. But in terms of doing the process correctly, it was a success.
Reflection:
This lab was an effective one in that my group really worked well and got the lab done in a timely manner. Also, my group didn't make fun of me like the last group, so that's a plus. But I could have done more work overall in this lab and been a more effective lab partner. At first this lab was confusing, but it got better as the lab went on. Overall, this was one of the best labs I have ever did this year so far.
3. The red colonies would have only appeared on the araganose because it allows the red protein to be expressed.
4.The more copies there are, the more products there are.
5. The central dogma (DNA- RNA- Protein) ends with a protein. The RFP protein is expressed in the form of the red glow as a trait.
6. All genes have the same central dogma.
Lab 6a:
Pre-Lab Question:
1. Proteins are unfolded in the highly salt concentrated buffer. The proteins that don't flow out the column are refolded when lower salt concentrations are added, so they leave the column.
Post-Lab Questions:
1. It determines the promoter regions of the given protein. These promoter regions are what give each protein their different functions.
2. The sequence determines how it will fold in a protein
3. The RFP-containing elute is brighter than the cell lysate because the elute contains the most RFP while the cell lysate contained all the cell proteins.
4. The red fluorescent protein sticks to the resin column when unfolded
5. To increase the purity of the red fluorescent protein sample, we could use more wash buffers and be more careful about collection.
Analysis/ Conclusion:
In this lab, we tried to make a red fluorescent bacteria colony. But we could not make it glow red because we got faulty material so it wasn't any of our fault. We still were able to make the protein but not use it for what it was supposed to. So in terms of actually getting it to work, we failed. But in terms of doing the process correctly, it was a success.
Reflection:
This lab was an effective one in that my group really worked well and got the lab done in a timely manner. Also, my group didn't make fun of me like the last group, so that's a plus. But I could have done more work overall in this lab and been a more effective lab partner. At first this lab was confusing, but it got better as the lab went on. Overall, this was one of the best labs I have ever did this year so far.