RFP lab
1.Purpose- Make RFP from jelly fish in bacteria
- Learn about steps genetic engineering
2.Materials + Procedure
1.Purpose- Make RFP from jelly fish in bacteria
- Learn about steps genetic engineering
2.Materials + Procedure
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
3.Experimental Overview:
Part 2a: Verification of plasmid by restriction digest - cut plasmid with BamHI and Hind III to cut out RFP-ara from bacterial plasmid.
Part 4a: Verification of plasmid digest by electrophoresis.
Part 5a: Transformation of bacteria with recombinant plasmid.The relevant components on the plasmid are the red fluorescent protein (rfp) gene, the promoter (pBAD), the ampicillin resistance gene (ampR), and the arabinose activator (araC).
Part 6: Portification of RFP using chromatography.
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.
The two fragments are RFP with pBAD and Ara-C with ori with Amp-R. The RFP and pBAD is 807 BP, while 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 which binds to the promoter.
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 is crucial because it determines the amount of bacteria that will grow and live while the other bacteria will die. This separates the bacteria into portions we need.
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?
They are defense mechanisms. By cutting the DNA out of other bacteria, they die giving bacteria a better chance to retain nutrients from soil.
3. Using your understanding of evolution, why would bacteria retain a genethat gives them resistance to antibiotics? How is the existence of bacteriawith antibiotic resistance affecting medicine today?
This allows bacteria not to die off. If bacteria gains resistance, antibiotics won't work, killing us from diseases and illnesses that were harmful 300 years ago like an ear infection.
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.
All follow the same central dogma DNA to mRNA through RNA polymerase, and mRNA to a protein through ribosomes.
5. Due to a mishap in the lab, bacteria carrying a plasmid with an ampicillinresistant 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.
Put Amp and Kan into a petri dish resistant to one. Repeat in another petri dish resistant to the other one. Once they are separated, we put them in different test tubes.
4a Questions:
1. The purpose of growing bacteria with ampicillin is that they are resistant and will not die. The p-Ara-R plasmid controls the cell's growth. Ampicillin kills unwanted bacteria.
2. You cannot control the production of your protein of interest since transcription cannot occur when the promoter isn't turned on by arabinose. RFP could not be expressed (no red).
3. I predicted the P+ in the LB plate will grow most abundant since it is the most inhabitual environment and P+ contains competent cells which support bacteria growth. Only cells with plasmids will grow.
5a Questions:
1. Our predictions were correct. The LB plate grew the most on it. The bacteria grew on both sides of the amp-ara, and on the LB-Amp, bacteria only grew on the P+ side but not the P- side.
2. There were no red colonies present either due to temperature or not enough time in the incubator.
3. The LB-amp-ara plate inhibits transcription to occur, which allows RFP to be expressed. The LB-amp does not have ara, a sugar.
4. It is important to have multiple copies because you can have extras for cell growth. Also, you can make more proteins.
5. The RFP gene is expressed as a trait through transcription from DNA to mRNA to protein (central dogma). This can happen because of ara-C.
6. Bacteria can again use transcription in the central dogma, and it can become a protein since it is resistant to different viruses, diseases, etc. Bacteria can make protein like humans because we both go through the same transcription.
6a Questions:
1. The red fluorescent protein can be depicted in each separation because the red cells will be at the bottom.
2. The supernatant was a clear tinted red liquid. The pellet is pink. The content was 150 microliters of EB and 150 microliters of LyB, along with red cells and bacteria.
6b Questions:
1. Binding Buffer (BB): create condition where amino acid and protein bind to the resin beads.
Wash Buffer (WB): washes off any proteins that are not tightly bound to the resin beads.
Elution Buffer (EB): high salt buffer, knocks protein off resin beads.
Column Equilibration Buffer (CEB): nice condition of salt and pH to store resin beads.
2. The supernatant was pinker than the last time. The pellet was only slightly darker pink than the supernatant. The content was 150 microliters of EB and 150 microliters of LyB, along with red cells and bacteria.
Analysis and Conclusion
In the lab we forgot the ARA promoter, which made the colonies not red. We redid it with bacteria that was red and grew the colonies from there. My group results came out as we expected and we were able to have our colonies.
Gel Analysis
Our class' gels failed to show the RFP, so we borrowed gels from another class and we're going to analyze them. The lane I'm analyzing is the last one to the right. This lane is pretty pure, but not completely because there's traces of other proteins in the gel. This is most likely due to when they were doing the chromatography they either didn't wash the tube completely or they accidentally collected some of the washing fluid with their RFP. The RFP is supposed to be 28 kD but their's is only about 17 kD.
Reflection
In this project I would of chose to have a different group. My group members were very closed up and wanted to do stuff themselves. I do believe we worked well and got stuff done. I think that in the future by being more assertive I will be able to be more helpful in the group. Over all i think my group worked well together. I really enjoyed working with Jessica and Eric.
Next time I think by double checking and understanding the lab more would be more efficient. Also, if i had known how to use all the tools i would have been able to be more helpful, but my group used a chromatography which i had to learn to use. It was quiet simply, yet frustrating.
I really enjoyed this project the chromatography machine had to have been my favorite part using it was so easy compared to what the other kids had to use in the class. It was a new machine that was donated to our school, and only few people were taught to use it and I am happy I was one.
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
3.Experimental Overview:
Part 2a: Verification of plasmid by restriction digest - cut plasmid with BamHI and Hind III to cut out RFP-ara from bacterial plasmid.
Part 4a: Verification of plasmid digest by electrophoresis.
Part 5a: Transformation of bacteria with recombinant plasmid.The relevant components on the plasmid are the red fluorescent protein (rfp) gene, the promoter (pBAD), the ampicillin resistance gene (ampR), and the arabinose activator (araC).
Part 6: Portification of RFP using chromatography.
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.
The two fragments are RFP with pBAD and Ara-C with ori with Amp-R. The RFP and pBAD is 807 BP, while 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 which binds to the promoter.
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 is crucial because it determines the amount of bacteria that will grow and live while the other bacteria will die. This separates the bacteria into portions we need.
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?
They are defense mechanisms. By cutting the DNA out of other bacteria, they die giving bacteria a better chance to retain nutrients from soil.
3. Using your understanding of evolution, why would bacteria retain a genethat gives them resistance to antibiotics? How is the existence of bacteriawith antibiotic resistance affecting medicine today?
This allows bacteria not to die off. If bacteria gains resistance, antibiotics won't work, killing us from diseases and illnesses that were harmful 300 years ago like an ear infection.
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.
All follow the same central dogma DNA to mRNA through RNA polymerase, and mRNA to a protein through ribosomes.
5. Due to a mishap in the lab, bacteria carrying a plasmid with an ampicillinresistant 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.
Put Amp and Kan into a petri dish resistant to one. Repeat in another petri dish resistant to the other one. Once they are separated, we put them in different test tubes.
4a Questions:
1. The purpose of growing bacteria with ampicillin is that they are resistant and will not die. The p-Ara-R plasmid controls the cell's growth. Ampicillin kills unwanted bacteria.
2. You cannot control the production of your protein of interest since transcription cannot occur when the promoter isn't turned on by arabinose. RFP could not be expressed (no red).
3. I predicted the P+ in the LB plate will grow most abundant since it is the most inhabitual environment and P+ contains competent cells which support bacteria growth. Only cells with plasmids will grow.
5a Questions:
1. Our predictions were correct. The LB plate grew the most on it. The bacteria grew on both sides of the amp-ara, and on the LB-Amp, bacteria only grew on the P+ side but not the P- side.
2. There were no red colonies present either due to temperature or not enough time in the incubator.
3. The LB-amp-ara plate inhibits transcription to occur, which allows RFP to be expressed. The LB-amp does not have ara, a sugar.
4. It is important to have multiple copies because you can have extras for cell growth. Also, you can make more proteins.
5. The RFP gene is expressed as a trait through transcription from DNA to mRNA to protein (central dogma). This can happen because of ara-C.
6. Bacteria can again use transcription in the central dogma, and it can become a protein since it is resistant to different viruses, diseases, etc. Bacteria can make protein like humans because we both go through the same transcription.
6a Questions:
1. The red fluorescent protein can be depicted in each separation because the red cells will be at the bottom.
2. The supernatant was a clear tinted red liquid. The pellet is pink. The content was 150 microliters of EB and 150 microliters of LyB, along with red cells and bacteria.
6b Questions:
1. Binding Buffer (BB): create condition where amino acid and protein bind to the resin beads.
Wash Buffer (WB): washes off any proteins that are not tightly bound to the resin beads.
Elution Buffer (EB): high salt buffer, knocks protein off resin beads.
Column Equilibration Buffer (CEB): nice condition of salt and pH to store resin beads.
2. The supernatant was pinker than the last time. The pellet was only slightly darker pink than the supernatant. The content was 150 microliters of EB and 150 microliters of LyB, along with red cells and bacteria.
Analysis and Conclusion
In the lab we forgot the ARA promoter, which made the colonies not red. We redid it with bacteria that was red and grew the colonies from there. My group results came out as we expected and we were able to have our colonies.
Gel Analysis
Our class' gels failed to show the RFP, so we borrowed gels from another class and we're going to analyze them. The lane I'm analyzing is the last one to the right. This lane is pretty pure, but not completely because there's traces of other proteins in the gel. This is most likely due to when they were doing the chromatography they either didn't wash the tube completely or they accidentally collected some of the washing fluid with their RFP. The RFP is supposed to be 28 kD but their's is only about 17 kD.
Reflection
In this project I would of chose to have a different group. My group members were very closed up and wanted to do stuff themselves. I do believe we worked well and got stuff done. I think that in the future by being more assertive I will be able to be more helpful in the group. Over all i think my group worked well together. I really enjoyed working with Jessica and Eric.
Next time I think by double checking and understanding the lab more would be more efficient. Also, if i had known how to use all the tools i would have been able to be more helpful, but my group used a chromatography which i had to learn to use. It was quiet simply, yet frustrating.
I really enjoyed this project the chromatography machine had to have been my favorite part using it was so easy compared to what the other kids had to use in the class. It was a new machine that was donated to our school, and only few people were taught to use it and I am happy I was one.