RFP (Red FLUORESCENT PROTEIN) Lab
Purpose: Make RFP from Jelly fish in Bacteria and to Learn about the steps of genetic engineering.
Materials and Procedures
Lab 2a: Materials and Procedure are in the Amgen Biotech experience lab part 2a
Lab 4a: Materials and Procedure are in the Amgen Biotech experience lab part 4a
Lab 5a: Materials and Procedure are in the Amgen Biotech experience lab part 5a
Lab 6: Materials and Procedure are in the Amgen Biotech experience lab part 6a
Lab 2a: Materials and Procedure are in the Amgen Biotech experience lab part 2a
Lab 4a: Materials and Procedure are in the Amgen Biotech experience lab part 4a
Lab 5a: Materials and Procedure are in the Amgen Biotech experience lab part 5a
Lab 6: Materials and Procedure are in the Amgen Biotech experience lab part 6a
Experimental Overview
Part 2a: Verification of plasmid by restriction digest We cut out the bacterial plasmid with BanH I and Hind III to cut out the RFP-ara from the bacterial plasmid.
Part 4a: We verified that we got the plasmid digest by using electrophoresis.
Part 5a: We transformed the the bacteria with the recombinant plasmid.
Part 6: We purified the RFP using chromatography.
Part 2a: Verification of plasmid by restriction digest We cut out the bacterial plasmid with BanH I and Hind III to cut out the RFP-ara from the bacterial plasmid.
Part 4a: We verified that we got the plasmid digest by using electrophoresis.
Part 5a: We transformed the the bacteria with the recombinant plasmid.
Part 6: We purified the RFP using chromatography.
Results:
Lab 2a We cut the solution with restriction enzymes and then ran it through a gel in lab 4a we did this to see how the restriction enzyme would work and to test it
Lab 4a We made the gel and ran it.
Results below the gel of 2a and 4a
Lab 2a We cut the solution with restriction enzymes and then ran it through a gel in lab 4a we did this to see how the restriction enzyme would work and to test it
Lab 4a We made the gel and ran it.
Results below the gel of 2a and 4a
Lab 5a We put the different substances on petrie dish plates to grow, but the plate were incorrectly made so the RFP was out and did not show.
Lab 6 We acquired protein that had the RFP dominant and ran it through a chromatography column and anylzed it to prove that the steps we did were correct and that we succeeded. Then we used electrophoresis to prove it.
The gel was pure but not extremely because there were many bands. The reason it might not be pure because we used the chromatography tubes wrong and made an error.
More data in the gel below
Lab 6 We acquired protein that had the RFP dominant and ran it through a chromatography column and anylzed it to prove that the steps we did were correct and that we succeeded. Then we used electrophoresis to prove it.
The gel was pure but not extremely because there were many bands. The reason it might not be pure because we used the chromatography tubes wrong and made an error.
More data in the gel below
Lab Questions
Pre-lab Q's
1. We cut the plasmid into two pieces one had the RFP with pBAD that was 805 base pairs the Other piece had ori ARA-c and ARA-r that had 4495 base pairs
2. RFP and ARA-c are needed to create the plasmid
3. A select able marker is needed to clone a gene because if it didn't have it, it would die, and separate it from the other DNA so it can grow.
Lab 2a Q's
1. Ori - origin, RFP - red fluorescent protein, AMP-r - selective marker, AMA-c Binds to the promoter to make transcription happen.
2. Restriction Enzymes fight off unknown bodies as a defense
3. Bacteria would retain a gene to survive. In medicine today it is bad for bacteria to retain genes because if the cure dosent work you have to find another and it is not good.
4. All organisms share the same central dogma
5. Have a petri dish that contains the Kanamycin. spread the AMP bacteria on only half of the dish. The bacteria should have died and you should be able to separate the 2 substances.
Lab 4a Q's
1. Ampicillin- it lets only certain call to grow
2. With out arabinose the pARA-r can connect to the promoter so nothing can turn red
3. LB plate: P-/P+ growth, LB/AMP Plate: P- restricted/P+ growth, LB/AMP/ARA Plate: Limited Bacteria Growth.
Lab 5a Q's
1. Our predictions were not clase at all we expected a lot of growth but nohing really happened on any of our plates
2. There weren't any red colonies at first we thought it wasn't at the right temp but then we found out the materials we got were bad
3. RFP is not expressed because LB/AMP/ARA prevented transcription and the LB/AMP plate didn't have ARA
4. More copies more of a chance of the promoter being activated
5. RFP gene is put on by transcription
Lab 6a Q's
1. RFP is separate because of its red cells
2. The super natant was very lite pink and the super and the pellet was lite pink
Lab 6b Q's
1. BB- Binding buffer- Causes binding between resin bed and the protein/amino acids
WB- wash buffer- removes excess protein
EB- Elution buffer- removes protein binds to the resin beads
CEB -Column Equilibrium Buffer- cleans resin beads
2. This time the super natant was lite pink and the pellet was a lot darker pink
Pre-lab Q's
1. We cut the plasmid into two pieces one had the RFP with pBAD that was 805 base pairs the Other piece had ori ARA-c and ARA-r that had 4495 base pairs
2. RFP and ARA-c are needed to create the plasmid
3. A select able marker is needed to clone a gene because if it didn't have it, it would die, and separate it from the other DNA so it can grow.
Lab 2a Q's
1. Ori - origin, RFP - red fluorescent protein, AMP-r - selective marker, AMA-c Binds to the promoter to make transcription happen.
2. Restriction Enzymes fight off unknown bodies as a defense
3. Bacteria would retain a gene to survive. In medicine today it is bad for bacteria to retain genes because if the cure dosent work you have to find another and it is not good.
4. All organisms share the same central dogma
5. Have a petri dish that contains the Kanamycin. spread the AMP bacteria on only half of the dish. The bacteria should have died and you should be able to separate the 2 substances.
Lab 4a Q's
1. Ampicillin- it lets only certain call to grow
2. With out arabinose the pARA-r can connect to the promoter so nothing can turn red
3. LB plate: P-/P+ growth, LB/AMP Plate: P- restricted/P+ growth, LB/AMP/ARA Plate: Limited Bacteria Growth.
Lab 5a Q's
1. Our predictions were not clase at all we expected a lot of growth but nohing really happened on any of our plates
2. There weren't any red colonies at first we thought it wasn't at the right temp but then we found out the materials we got were bad
3. RFP is not expressed because LB/AMP/ARA prevented transcription and the LB/AMP plate didn't have ARA
4. More copies more of a chance of the promoter being activated
5. RFP gene is put on by transcription
Lab 6a Q's
1. RFP is separate because of its red cells
2. The super natant was very lite pink and the super and the pellet was lite pink
Lab 6b Q's
1. BB- Binding buffer- Causes binding between resin bed and the protein/amino acids
WB- wash buffer- removes excess protein
EB- Elution buffer- removes protein binds to the resin beads
CEB -Column Equilibrium Buffer- cleans resin beads
2. This time the super natant was lite pink and the pellet was a lot darker pink
Conclusion and Reflection:
This was an interesting lab with many steps. In conclusion the RFP gene actually did not work because the plates and supplies we used did not work. After the experiment failed the teacher acquired the RFP that work and we got color change so the RFP gene was present. We analyzed it. This project was all about the purpose of biotech in the world. Also to show us the job of what bio technologists do everyday.
This lab was fun and interesting in many ways because we did almost everything in one lab. We restricted enzymes, cut enzymes, we made solutions, and did a lot of micropipeting. My group was awesome and we made a good time of it. One bad thing that happened was, I was the only one in my group who knew what was going on. Overall this lab was interesting, but labor intensive.
This was an interesting lab with many steps. In conclusion the RFP gene actually did not work because the plates and supplies we used did not work. After the experiment failed the teacher acquired the RFP that work and we got color change so the RFP gene was present. We analyzed it. This project was all about the purpose of biotech in the world. Also to show us the job of what bio technologists do everyday.
This lab was fun and interesting in many ways because we did almost everything in one lab. We restricted enzymes, cut enzymes, we made solutions, and did a lot of micropipeting. My group was awesome and we made a good time of it. One bad thing that happened was, I was the only one in my group who knew what was going on. Overall this lab was interesting, but labor intensive.