RFP And GEnetic Engineering
Purpose: Our reason for this lab was to create a RFP, that comes from Jellyfish, and place it into Bacteria. Through this process, we would learn the steps to Genetic Engineering
Procedure and Materials:
Lab 2A: The procedure and materials list can be found in the Amgen Biotech Experience Manuel part 2A
Lab 4A: The procedure and materials list can be found in the Amgen Biotech Experience Manuel part 4A
Lab 5A: The procedure and materials list can be found in the Amgen Biotech Experience Manuel part 5A
Lab 6A: The procedure and materials list can be found in the Amgen Biotech Experience Manuel part 6A
Experimental Overview:
Lab 2A: We verified that the DNA we had also acquired the plasmid we wanted by using restriction digest. We isolated the verified RFP-ara Bacteria by cutting it with BanH I and Hind III and then extracting the piece.
Lab 4A: We verified we had the plasmid digest by using electrophoresis.
Lab 5A: We then transformed the bacteria using a recombinant plasmid.
Lab 6A: We purified our plasmid using chromatography.
Results:
Pre-Lab 2A Questions:
1) We had produced 2 different fragments. One was an RFP combined with pBad and the other was ARA-c with ori as well as AMP-r. The first fragment had a total BP was 807 while the second fragment had a BP of 4495.
2) RFP and ARA-c are both necessary components of the plasmid.
3) A selectable marker can separate at bacteria from the rest of the gene and thus allow the bacteria to grow on its own.
Lab 2A Questions:
1) Ori - Origin of the Replication, RFP - Red Fluorescent Protein, Amp-r - Selectable Marker, ARA-c - It binds to the promoter and can create a transcription of the gene of interest.
2) Restriction Enzymes attack any foreign bodies as a means of defense.
3) Bacteria has genes that resist diseases as well as antibiotics.
4) All organisms follow 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 Questions:
1) Ampicillin-resistant bacteria can allow only a select amount of cells to grow.
2) With out the arabinose, the pARA-r plasmid can't activate the promoter which in turn prevents the protein from turning red.
3) LB plate: P-/P+ growth, LB/AMP Plate: P- restricted/P+ growth, LB/AMP/ARA Plate: Limited Bacteria Growth.
Lab 5A Questions:
1) Our predictions were as follows: The LB plate was close to our prediction. The LB/AMP and LB/AMP/ARA, on the other hand, did match up with our predictions.
2) The lack of red colonies could have been due to a limited time within the incubator or an incorrect temperature.
3) The RFP is expressed because the LB/AMP/ARA plate prevented transcription.The LB/AMP plate didn't contain the ARA.
4) With an increased amount of copies, the greater the chance of the promoter being activated.
5) The RFP Gene is a trait expressed by transcription.
Lab 6A Questions:
1) The RFP is separated due to its red cells
2) The supernatant was a clear colored liquid while the pellet was pink.
Lab 6B Questions:
1) Binding Buffer (BB): Causes a binding between the resin bed and the protein and amino acids
Wash Buffer (WB): Removes excess protein that isn't bound to the resin bed.
Elution Buffer (EB): Extracts the protein that is bound to the resin bed.
Column Equilibration Buffer (CEB): Stores resin beads.
2) This time around, the supernatant was pink while the pellet had become a darker pink.
Reflection:
The lab had gone well and with few problem, those that did occur were dealt with quickly. I felt that this lab, as a whole, was actually easier and more fun than our previous experiments. My skills have improved as the year has gone on. These improved skills are what allowed this lab to go the way it did. My group-mates and I accomplished what we had to do at a rate that definitely surprised me in a good way. I loved performing this activity and wouldn't mind doing it again.
Procedure and Materials:
Lab 2A: The procedure and materials list can be found in the Amgen Biotech Experience Manuel part 2A
Lab 4A: The procedure and materials list can be found in the Amgen Biotech Experience Manuel part 4A
Lab 5A: The procedure and materials list can be found in the Amgen Biotech Experience Manuel part 5A
Lab 6A: The procedure and materials list can be found in the Amgen Biotech Experience Manuel part 6A
Experimental Overview:
Lab 2A: We verified that the DNA we had also acquired the plasmid we wanted by using restriction digest. We isolated the verified RFP-ara Bacteria by cutting it with BanH I and Hind III and then extracting the piece.
Lab 4A: We verified we had the plasmid digest by using electrophoresis.
Lab 5A: We then transformed the bacteria using a recombinant plasmid.
Lab 6A: We purified our plasmid using chromatography.
Results:
Pre-Lab 2A Questions:
1) We had produced 2 different fragments. One was an RFP combined with pBad and the other was ARA-c with ori as well as AMP-r. The first fragment had a total BP was 807 while the second fragment had a BP of 4495.
2) RFP and ARA-c are both necessary components of the plasmid.
3) A selectable marker can separate at bacteria from the rest of the gene and thus allow the bacteria to grow on its own.
Lab 2A Questions:
1) Ori - Origin of the Replication, RFP - Red Fluorescent Protein, Amp-r - Selectable Marker, ARA-c - It binds to the promoter and can create a transcription of the gene of interest.
2) Restriction Enzymes attack any foreign bodies as a means of defense.
3) Bacteria has genes that resist diseases as well as antibiotics.
4) All organisms follow 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 Questions:
1) Ampicillin-resistant bacteria can allow only a select amount of cells to grow.
2) With out the arabinose, the pARA-r plasmid can't activate the promoter which in turn prevents the protein from turning red.
3) LB plate: P-/P+ growth, LB/AMP Plate: P- restricted/P+ growth, LB/AMP/ARA Plate: Limited Bacteria Growth.
Lab 5A Questions:
1) Our predictions were as follows: The LB plate was close to our prediction. The LB/AMP and LB/AMP/ARA, on the other hand, did match up with our predictions.
2) The lack of red colonies could have been due to a limited time within the incubator or an incorrect temperature.
3) The RFP is expressed because the LB/AMP/ARA plate prevented transcription.The LB/AMP plate didn't contain the ARA.
4) With an increased amount of copies, the greater the chance of the promoter being activated.
5) The RFP Gene is a trait expressed by transcription.
Lab 6A Questions:
1) The RFP is separated due to its red cells
2) The supernatant was a clear colored liquid while the pellet was pink.
Lab 6B Questions:
1) Binding Buffer (BB): Causes a binding between the resin bed and the protein and amino acids
Wash Buffer (WB): Removes excess protein that isn't bound to the resin bed.
Elution Buffer (EB): Extracts the protein that is bound to the resin bed.
Column Equilibration Buffer (CEB): Stores resin beads.
2) This time around, the supernatant was pink while the pellet had become a darker pink.
Reflection:
The lab had gone well and with few problem, those that did occur were dealt with quickly. I felt that this lab, as a whole, was actually easier and more fun than our previous experiments. My skills have improved as the year has gone on. These improved skills are what allowed this lab to go the way it did. My group-mates and I accomplished what we had to do at a rate that definitely surprised me in a good way. I loved performing this activity and wouldn't mind doing it again.
The Photo above shows the protein placed into a gel and put under Electrophoresis.