purpose
To express red fluorescent protein from jellyfish in bacteria. This expirement would also help us learn about genetic engineering.
Materials
Lab 2a
Materials can be found in Amgen lab manual part 2a.
Lab 4a
Materials can be found in Amgen lab manual part 4a.
Lab 5a
Materials can be found in Amgen lab manual part 5a.
Lab 6
Materials can be found in Amgen lab manual part 6.
Materials can be found in Amgen lab manual part 2a.
Lab 4a
Materials can be found in Amgen lab manual part 4a.
Lab 5a
Materials can be found in Amgen lab manual part 5a.
Lab 6
Materials can be found in Amgen lab manual part 6.
Overview
Lab 2a - We verified that we had the correct plasma by using a restriction digest. We cut the plasmid with BamHI and Hind III.
Lab 4a - We verified the plasmid digest by electrophoresis.
Lab 5a - Then we transformed the bacteria with a recombinant plasmid.
Lab 6 - The we purified the RFP using chromotography.
Lab 4a - We verified the plasmid digest by electrophoresis.
Lab 5a - Then we transformed the bacteria with a recombinant plasmid.
Lab 6 - The we purified the RFP using chromotography.
REsults
Before the 2a Lab:
1. If pARA-R is digested with BamHI and HindIII, what fragments are produced? Record the nucleotide sequence of the sticky ends and the length of each fragment (bp), and indicate the genes and other important sequences present on each fragment. Two fragments are produced. They are RFP with pBAD and Ara-C with ori with Amp-R.. The RFP plus pBAD is 807 BP, and Ara-C, ori, and Amp-R is 4495 BP.
2. In order to create a plasmid that can produce the red fluorescent protein in
bacteria, what components are needed in the plasmid? The RFP gene and Ara-Care the needed components in the plasmid .
3. If the uptake of DNA by bacteria is inefficient (as discussed in the reading), why is a selectable marker (gene with resistance to an antibiotic) critical in cloning a gene in bacteria? The selectable marker allows only the desired bacteria to grow. It separates the bacteria from the desired gene.
2a Questions:
1. List in words or indicate in a drawing the important features of a plasmid vector that are required to clone a gene. Explain the purpose of each feature. Ori = origin of replication; RFP = red fluorescent protein (gene of interest); Amp-R = selectable marker; Ara-C = binds to promoter so we get transcription of gene of interest.
2. What role do restriction enzymes have in nature? Restriction enzymes are a defense mechanism. They cut up foreign bodies.
3. 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 retain genes that give them resistance to antibiotics to protect themselves from disease.
4. Bacteria, sea anemones, and humans seem, on the surface, to be very different organisms. Explain how a gene from humans or a sea anemone can be expressed in bacteria to make a product never before made in bacteria. The central dogma is the same in all organisms.
5. 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. Create a petry dish that grows both Kan and Amp bacteria. Put half of the mixed bacteria into each petry dish and wait a day. The bacteria should have died and then you should be able to separate them apart.
4a Questions:
1. The purpose of growing bacteria that resists ampicillin is to make sure that the desired cell lives.
2. If the bacterial cells are not given arabinose, then the pARA-R plasmid will not turn on the promoter. Then the protein will not turn red.
3. In the LB plate, both the P- and P+ will grow. In the LB/Amp plate, no P- will grow, but P+ will. In the LB/ Amp/Ara plate, there will barely any bacteria growth.
5a Questions:
1. Our predictions sort of matched our results. The LB plate had limited growth. The LB/Amp and LB/Amp/Ara plate matched our prediction.
2. There were no red colonies visible either due to temperature or not enough time in the incubator.
3. The LB-amp-ara plate prevents transcription to occur, which allows RFP to be expressed. The LB-amp does not have Ara.
4. It is important to have multiple copies so there is a greater chance of the promoter being turned on.
5. The RFP gene is expressed as a trait through transcription (central dogma).
6a Questions:
1. The red fluorescent protein can be seen separated because of its red cells.
2. The supernatant is clear liquid. The pellet is pink.
6b Questions:
1. Binding Buffer (BB): causes amino acid and protein bind to the resin beads.
Wash Buffer (WB): removes loose proteins that are not bound to the resin beads.
Elution Buffer (EB): takes off protein off resin beads.
Column Equilibration Buffer (CEB): stores resin beads.
2. This time, the supernatant was more pink than clear. The pellet was a little darker pink than the supernatant.
1. If pARA-R is digested with BamHI and HindIII, what fragments are produced? Record the nucleotide sequence of the sticky ends and the length of each fragment (bp), and indicate the genes and other important sequences present on each fragment. Two fragments are produced. They are RFP with pBAD and Ara-C with ori with Amp-R.. The RFP plus pBAD is 807 BP, and Ara-C, ori, and Amp-R is 4495 BP.
2. In order to create a plasmid that can produce the red fluorescent protein in
bacteria, what components are needed in the plasmid? The RFP gene and Ara-Care the needed components in the plasmid .
3. If the uptake of DNA by bacteria is inefficient (as discussed in the reading), why is a selectable marker (gene with resistance to an antibiotic) critical in cloning a gene in bacteria? The selectable marker allows only the desired bacteria to grow. It separates the bacteria from the desired gene.
2a Questions:
1. List in words or indicate in a drawing the important features of a plasmid vector that are required to clone a gene. Explain the purpose of each feature. Ori = origin of replication; RFP = red fluorescent protein (gene of interest); Amp-R = selectable marker; Ara-C = binds to promoter so we get transcription of gene of interest.
2. What role do restriction enzymes have in nature? Restriction enzymes are a defense mechanism. They cut up foreign bodies.
3. 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 retain genes that give them resistance to antibiotics to protect themselves from disease.
4. Bacteria, sea anemones, and humans seem, on the surface, to be very different organisms. Explain how a gene from humans or a sea anemone can be expressed in bacteria to make a product never before made in bacteria. The central dogma is the same in all organisms.
5. 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. Create a petry dish that grows both Kan and Amp bacteria. Put half of the mixed bacteria into each petry dish and wait a day. The bacteria should have died and then you should be able to separate them apart.
4a Questions:
1. The purpose of growing bacteria that resists ampicillin is to make sure that the desired cell lives.
2. If the bacterial cells are not given arabinose, then the pARA-R plasmid will not turn on the promoter. Then the protein will not turn red.
3. In the LB plate, both the P- and P+ will grow. In the LB/Amp plate, no P- will grow, but P+ will. In the LB/ Amp/Ara plate, there will barely any bacteria growth.
5a Questions:
1. Our predictions sort of matched our results. The LB plate had limited growth. The LB/Amp and LB/Amp/Ara plate matched our prediction.
2. There were no red colonies visible either due to temperature or not enough time in the incubator.
3. The LB-amp-ara plate prevents transcription to occur, which allows RFP to be expressed. The LB-amp does not have Ara.
4. It is important to have multiple copies so there is a greater chance of the promoter being turned on.
5. The RFP gene is expressed as a trait through transcription (central dogma).
6a Questions:
1. The red fluorescent protein can be seen separated because of its red cells.
2. The supernatant is clear liquid. The pellet is pink.
6b Questions:
1. Binding Buffer (BB): causes amino acid and protein bind to the resin beads.
Wash Buffer (WB): removes loose proteins that are not bound to the resin beads.
Elution Buffer (EB): takes off protein off resin beads.
Column Equilibration Buffer (CEB): stores resin beads.
2. This time, the supernatant was more pink than clear. The pellet was a little darker pink than the supernatant.
Reflection
I really liked this project because we did an experiment with materials that is applicable to what real biotech companies actually do, and we used materials they actually use. I also really liked working with the group that was chosen for me, we worked well together and all did equal parts and good work. Our biggest downfall was not staying on task, but regardless we had enough focus when we were to get everything done. Overall i think our group did very well.