Red Floresent Protien Lab
Purpose: Make RFP from jelly fish in bacteria and learn about steps of genetic engineering.
Materials and Procedures:
Experimental overview:
2a restriction digest to verify the plasmid
4a verify plasmid digest by electrophoresis
5a transport bacteria with recombinant plasmid
6 porification of RFP using chromotography
Results:
1. 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. There are 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? We need the RFP gene and Ara-C.
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.
5a Before the Lab Questions:
1. Ampicillin is an antibiotic that kills bacterial cells by disrupting the formation of cell walls. However, the pARA-R plasmid has the ampicillin resistance gene, which produces a protein that breaks down ampicillin. What is the purpose of growing bacteria that have been transformed in the presence of ampicillin?
The purpose of growing bacteria that resists ampicillin is to make sure that the desired cell lives.
2. What will happen when bacterial cells that contain the pARA-R plasmid are not given arabinose?
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 lab, you will add samples of the control group P– and the treatment group P+ to plates that contain various combinations of Luria Broth (LB), ampicillin, and the sugar arabinose. The plates will be arranged as follows:
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.In the lab, you will add samples of the control group P– and the treatment group P+ to plates that contain various combinations of Luria Broth (LB), ampicillin, and the sugar arabinose. The plates will be arranged as follows:
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. How many red colonies were present on your LB/amp/ara plate?
There were no red colonies visible either due to temperature or not enough time in the incubator.
3. Why did the red colonies only appear on the LB/amp/ara plate and not the
LB/amp plate?
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).
6.
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
Materials and Procedures:
- 2a - materials and procedure in Amgen lab manual part 2a
- 4a - materials and procedure in Amgen lab manual part 4a
- 5a - materials and procedure in Amgen lab manual part 5a
- 6 - materials and procedure in Amgen lab manual part 6
Experimental overview:
2a restriction digest to verify the plasmid
4a verify plasmid digest by electrophoresis
5a transport bacteria with recombinant plasmid
6 porification of RFP using chromotography
Results:
1. 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. There are 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? We need the RFP gene and Ara-C.
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.
5a Before the Lab Questions:
1. Ampicillin is an antibiotic that kills bacterial cells by disrupting the formation of cell walls. However, the pARA-R plasmid has the ampicillin resistance gene, which produces a protein that breaks down ampicillin. What is the purpose of growing bacteria that have been transformed in the presence of ampicillin?
The purpose of growing bacteria that resists ampicillin is to make sure that the desired cell lives.
2. What will happen when bacterial cells that contain the pARA-R plasmid are not given arabinose?
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 lab, you will add samples of the control group P– and the treatment group P+ to plates that contain various combinations of Luria Broth (LB), ampicillin, and the sugar arabinose. The plates will be arranged as follows:
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.In the lab, you will add samples of the control group P– and the treatment group P+ to plates that contain various combinations of Luria Broth (LB), ampicillin, and the sugar arabinose. The plates will be arranged as follows:
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. How many red colonies were present on your LB/amp/ara plate?
There were no red colonies visible either due to temperature or not enough time in the incubator.
3. Why did the red colonies only appear on the LB/amp/ara plate and not the
LB/amp plate?
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).
6.
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:
The thing I liked most about this project was that we were actually able to see our results and that our results came out how we expected. A lot of time I science there experimental error that mess up our result so it was nice that it didnt this time. My group an I collaborated well, we all new each other and were able to stay on task. If I could do one thing different in this lab it would be asking more questions because I find myself all to often doing something but have o idea what it does or why im doing it.
The thing I liked most about this project was that we were actually able to see our results and that our results came out how we expected. A lot of time I science there experimental error that mess up our result so it was nice that it didnt this time. My group an I collaborated well, we all new each other and were able to stay on task. If I could do one thing different in this lab it would be asking more questions because I find myself all to often doing something but have o idea what it does or why im doing it.