Red Fluorescent protein Lab
Purpose
1. Make Red Fluorescent Protein from jelly fish in bacteria
2. Learn about the steps of genetic engineering
Materials and Procedure
2a: materials and procedure can be found in Amgen lab manual part 2A
4a: materials and procedure can be found in Amgen lab manual part 4A
5a: materials and procedure can be found in Amgen lab manual part 5A
6: materials and procedure can be found in Amgen lab manual part 6A
Experimental Overview
Part 2a: In this part we used BamH I and Hind III to cut out RFP-ara from the bacterial plasmid.
Part 4a: We used electrophoresis for verification of plasmid digest.
Part 5a: We used transformation of bacteria with recombinant plasmid.
Part 6: We purified the RFP using chromotography.
Data
2a before the lab:
1.The two fragments are RFP with pBAD and Ara-C with ori with Amp-R. The RFP and pBAD is 807 Base Pairs, while Ara-C, ori, and Amp-R is 4495 Base Pairs.
2. The components needed in the plasmid are Ara-C and the RFP gene.
3. The selectable marker is needed to determine which bacteria will survive.
2a Stop and Think:
1. The purpose of setting up the tube without the enzymes is to use it as a control sample.
2. The enzymes might work best at 37*C because that is the way it works in the human body so they are comfortable.
2a Questions:
1. Ori = origin of replication; RFP = red fluorescent protein (gene of interest); Amp-R = selectable marker; Ara-C = binds to promoter
2. The restriction enzymes act as a sort of defense weapon.
3. Bacteria would retain certain enzymes to allow them to live. It is making it more difficult in medicine today because the bacteria have become resistant and it is harder to kill them.
4. All follow the same central dogma DNA to mRNA through RNA polymerase, and mRNA to a protein.
5. Put ampicillin and Kanamycin on a petri dish. The resistant ones will survive on each side leaving them separated.
4a before the lab:
1. The plasmid may have been supercoiled.
4a Stop and Think:
1. The loading dye is useful because it allows you to see the DNA as it travels through the gel.
2. The DNA may be visible by using a loading dye.
4a Questions:
1. It is important because you want to make sure it will do what you want it to do.
2. My gel results were similar to my predictions.
3. I did not know that there would be a faint band in one of the lanes.
4. The gel does show that we are using the correct plasmid because the base pairs match up.
5. No because there is only one band.
6. In the R+ lane there are 2 bands.
7. You would expect to find the RFP gene in the R+ lane.
8. The DNA ladder did not show up so we were unable to compare the two.
5a before the lab:
1. Growing these ampicillin bacteria could lead to new understandings of medicines.
2. When not given arabinose, these bacteria would die.
3. I predict that the bacteria will grow on both sides of the amp- ara, and only the P+ will grow on the LB-amp.
5a Stop and Think:
1. The purpose of the P- is to be used as a control.
2. The cells need time to recover so that they don't die from the extreme temperatures.
3. Cells are incubated at 37*C because that is the temperature of the human body.
4. You use aseptic techniques so that you won't transfer any bacteria.
5a Questions:
1. Our results matched our predictions.
2. We did not get any red colonies however we did get colonies.
3. The colonies were not resistant to the LB-amp plate.
4. It is important to have multiple copies because you can have extras for cell growth.
5. The RFP gene is expressed as a trait through transcription from DNA to mRNA to protein.
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.
6 before the lab:
1. Salt solutions can be used to help the protein stick to the beads and help flush it off the beads.
6 Stop and Think:
1. You can determine where the RFP is because it has a reddish pink color.
2. The supernatant is clear or colorless. The pellet is pink.
3. The BB helps the protein to stick to the beads. The WB gets rid of everything that is not stuck on the beads. The EB flushes the protein.
6 Questions:
1. The eluate containing the RFP gene seemed less bright.
2. The color of the RFP was used as the basis for separation.
3. The precision of the chromatography column could be increased by using the machine which measures everything exactly, or by practicing more.
Conclusion/Analysis
When we originally did this project, our some bacteria colonies grew on our petri dishes, but they did not appear to be red. Later we found out the company gave us the wrong plasmids. When we did part 6 of the lab we were using the correct bacteria with the Red Fluorescent Protein. We were able to see the reddish pink color while separating it into the Pellet and Supernatant by using the centrifuge. We used the chromatography column to separate out the Red Fluorescent Protein which appeared pink.
We used the protein that we purified using the chromatography column in a vertical gel. There is a picture of it below in the slide show. If you look at it you can see that some came out more pure than others by looking at the number of bands. Our classes gels did not run very well , so the picture is of the other class's gel.
1. Make Red Fluorescent Protein from jelly fish in bacteria
2. Learn about the steps of genetic engineering
Materials and Procedure
2a: materials and procedure can be found in Amgen lab manual part 2A
4a: materials and procedure can be found in Amgen lab manual part 4A
5a: materials and procedure can be found in Amgen lab manual part 5A
6: materials and procedure can be found in Amgen lab manual part 6A
Experimental Overview
Part 2a: In this part we used BamH I and Hind III to cut out RFP-ara from the bacterial plasmid.
Part 4a: We used electrophoresis for verification of plasmid digest.
Part 5a: We used transformation of bacteria with recombinant plasmid.
Part 6: We purified the RFP using chromotography.
Data
2a before the lab:
1.The two fragments are RFP with pBAD and Ara-C with ori with Amp-R. The RFP and pBAD is 807 Base Pairs, while Ara-C, ori, and Amp-R is 4495 Base Pairs.
2. The components needed in the plasmid are Ara-C and the RFP gene.
3. The selectable marker is needed to determine which bacteria will survive.
2a Stop and Think:
1. The purpose of setting up the tube without the enzymes is to use it as a control sample.
2. The enzymes might work best at 37*C because that is the way it works in the human body so they are comfortable.
2a Questions:
1. Ori = origin of replication; RFP = red fluorescent protein (gene of interest); Amp-R = selectable marker; Ara-C = binds to promoter
2. The restriction enzymes act as a sort of defense weapon.
3. Bacteria would retain certain enzymes to allow them to live. It is making it more difficult in medicine today because the bacteria have become resistant and it is harder to kill them.
4. All follow the same central dogma DNA to mRNA through RNA polymerase, and mRNA to a protein.
5. Put ampicillin and Kanamycin on a petri dish. The resistant ones will survive on each side leaving them separated.
4a before the lab:
1. The plasmid may have been supercoiled.
4a Stop and Think:
1. The loading dye is useful because it allows you to see the DNA as it travels through the gel.
2. The DNA may be visible by using a loading dye.
4a Questions:
1. It is important because you want to make sure it will do what you want it to do.
2. My gel results were similar to my predictions.
3. I did not know that there would be a faint band in one of the lanes.
4. The gel does show that we are using the correct plasmid because the base pairs match up.
5. No because there is only one band.
6. In the R+ lane there are 2 bands.
7. You would expect to find the RFP gene in the R+ lane.
8. The DNA ladder did not show up so we were unable to compare the two.
5a before the lab:
1. Growing these ampicillin bacteria could lead to new understandings of medicines.
2. When not given arabinose, these bacteria would die.
3. I predict that the bacteria will grow on both sides of the amp- ara, and only the P+ will grow on the LB-amp.
5a Stop and Think:
1. The purpose of the P- is to be used as a control.
2. The cells need time to recover so that they don't die from the extreme temperatures.
3. Cells are incubated at 37*C because that is the temperature of the human body.
4. You use aseptic techniques so that you won't transfer any bacteria.
5a Questions:
1. Our results matched our predictions.
2. We did not get any red colonies however we did get colonies.
3. The colonies were not resistant to the LB-amp plate.
4. It is important to have multiple copies because you can have extras for cell growth.
5. The RFP gene is expressed as a trait through transcription from DNA to mRNA to protein.
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.
6 before the lab:
1. Salt solutions can be used to help the protein stick to the beads and help flush it off the beads.
6 Stop and Think:
1. You can determine where the RFP is because it has a reddish pink color.
2. The supernatant is clear or colorless. The pellet is pink.
3. The BB helps the protein to stick to the beads. The WB gets rid of everything that is not stuck on the beads. The EB flushes the protein.
6 Questions:
1. The eluate containing the RFP gene seemed less bright.
2. The color of the RFP was used as the basis for separation.
3. The precision of the chromatography column could be increased by using the machine which measures everything exactly, or by practicing more.
Conclusion/Analysis
When we originally did this project, our some bacteria colonies grew on our petri dishes, but they did not appear to be red. Later we found out the company gave us the wrong plasmids. When we did part 6 of the lab we were using the correct bacteria with the Red Fluorescent Protein. We were able to see the reddish pink color while separating it into the Pellet and Supernatant by using the centrifuge. We used the chromatography column to separate out the Red Fluorescent Protein which appeared pink.
We used the protein that we purified using the chromatography column in a vertical gel. There is a picture of it below in the slide show. If you look at it you can see that some came out more pure than others by looking at the number of bands. Our classes gels did not run very well , so the picture is of the other class's gel.
Reflection
For this lab, Dr. LB chose our groups for us. I worked with Bronte and Cate. It was actually quite nice working with people that I had never worked with. I think it was good to experience different peoples strengths and work ethics. We worked well and efficiently. One thing we did very well was dividing the work and communication about what needed to get done. One thing we need to work on is our pipetting. We actually used the wrong pipet at one point, when we were trying to grow our colonies. Luckily we had just enough to put some on each plate. I personally really likes seeing all of the colonies that had grown, even though they hadn't turned out red. I learned to have patience while we were using the chromatography column, because we had to wait for gravity to do its job.
For this lab, Dr. LB chose our groups for us. I worked with Bronte and Cate. It was actually quite nice working with people that I had never worked with. I think it was good to experience different peoples strengths and work ethics. We worked well and efficiently. One thing we did very well was dividing the work and communication about what needed to get done. One thing we need to work on is our pipetting. We actually used the wrong pipet at one point, when we were trying to grow our colonies. Luckily we had just enough to put some on each plate. I personally really likes seeing all of the colonies that had grown, even though they hadn't turned out red. I learned to have patience while we were using the chromatography column, because we had to wait for gravity to do its job.