RFP lab
Purpose: for this lab, we had two main purposes.
Lab 2a: Materials and Procedure are in the Amgen Biotech experience lab part 2a
Lab 4a: Materials and Procedure are in the Amgen Biotech experience lab part 4a
Lab 5a: Materials and Procedure are in the Amgen Biotech experience lab part 5a
Lab 6: Materials and Procedure are in the Amgen Biotech experience lab part 6a
Experimental Overview:
2a: we verified the plasmid by using the restriction digest We cut out the bacterial plasmid with BanH I and Hind III to cut out the RFP-ara from the bacterial plasmid.
4a: we then use electropheresis to verify the plasmid digest.
5a: We used recombinant plasmid to transform the bacteria.
6: Using the process of chromotography, we purified the RFP.
Questions:
before 2a:
- Make RFP from jelly fish in bacteria
- learn about the steps to genetic engineering.
Lab 2a: Materials and Procedure are in the Amgen Biotech experience lab part 2a
Lab 4a: Materials and Procedure are in the Amgen Biotech experience lab part 4a
Lab 5a: Materials and Procedure are in the Amgen Biotech experience lab part 5a
Lab 6: Materials and Procedure are in the Amgen Biotech experience lab part 6a
Experimental Overview:
2a: we verified the plasmid by using the restriction digest We cut out the bacterial plasmid with BanH I and Hind III to cut out the RFP-ara from the bacterial plasmid.
4a: we then use electropheresis to verify the plasmid digest.
5a: We used recombinant plasmid to transform the bacteria.
6: Using the process of chromotography, we purified the RFP.
Questions:
before 2a:
- There are two fragments produced, the RFP + PBAD and Ara-C + ori + Amp-R. The RFP and PBAD equal 807 BP when subtracted from each other, and Ara-C, ori, and Amp-R equal 4495 BP when subtracted from each other.
- The two genes that are needed are the RFP and Ara-c genes.
- the marker identifies the bacteria we want and separates the one without the desired gene.
- Ori- origin, where replication begins
- RFP- gene of interest
- Amp-R -provides resistance against antibiotics, acts as a selective marker
- Ara-C - binds to the promoter
- it acts as a defense mechanism by cutting up foreign DNA
- it would die off in a hostile environment because we need stronger and more advanced antibiotics.
- all living things have the same central dogma
- Take half of the mixture and put each half in a petri dish. Put the Amp gene in one of the dishes and the Kan gene in the other so the kan kills the amp gene and the amp kills the kan gene. This separates both Amp and Kan from each other.
- bacteria that resists ampicillin is made so the amp-r cells will survive.
- without arabinose, they wouldn't turn red.
- on the LB plate, p- and p+ will grow, while on the lb/amp plate, only P+ will grow, and a very little amount of bacteria will grow on the LB/Amp/Ara plate
5a:
1a. We can see and separate the RFP because it was red and fluorescent
2a. The supernatant was clear and the pellet had a little bit of a pink/red tint
1b.Binding Buffer - makes amino acid and protein bind to the resin beads.
Wash Buffer - removes the proteins not bound to resin beads
Elution Buffer - takes protein off the resin beads
Column Equilibration Buffer - stores the resin beads
2b. the supernatant was clear pink, while the pellet was a darker shade of red.
Data analysis/conclusion: In the end of this lab, me and my group were not able to create a red fluorescent protein. Although, we were able to participate in all the class procedures for this lab, such as isolation, electrophoresis, transferring, and purifying the RFP. We did all this while learning about bio engineering, so I guess there is learning in failure.
Reflection: This lab was complicated, long, and took some expertise, like a pizza buffet. I had fun during this lab, although being very, and i mean VERY, confused at times. I found the fact that we could the color of bacteria to be very interesting, considering that's magician stuff. Me and group worked well together.I would have liked if me and my group payed attention to the directions better, while also working more efficiently. But all in all, it was a fairly interesting experience that i would do again if i could, like eating a pizza-burrito.
Unfortunately, since we made mistakes along the way, we didn't end up with a gel or results.
- No, since we weren't able to do this part of the lab since we forgot to do a step early on.
- There were no red colonies
- Because the area assesses the RFP
- So they can interact and grow
- It doesn't
- Because they can isolate the gen and put it in some bacteria.
1a. We can see and separate the RFP because it was red and fluorescent
2a. The supernatant was clear and the pellet had a little bit of a pink/red tint
1b.Binding Buffer - makes amino acid and protein bind to the resin beads.
Wash Buffer - removes the proteins not bound to resin beads
Elution Buffer - takes protein off the resin beads
Column Equilibration Buffer - stores the resin beads
2b. the supernatant was clear pink, while the pellet was a darker shade of red.
Data analysis/conclusion: In the end of this lab, me and my group were not able to create a red fluorescent protein. Although, we were able to participate in all the class procedures for this lab, such as isolation, electrophoresis, transferring, and purifying the RFP. We did all this while learning about bio engineering, so I guess there is learning in failure.
Reflection: This lab was complicated, long, and took some expertise, like a pizza buffet. I had fun during this lab, although being very, and i mean VERY, confused at times. I found the fact that we could the color of bacteria to be very interesting, considering that's magician stuff. Me and group worked well together.I would have liked if me and my group payed attention to the directions better, while also working more efficiently. But all in all, it was a fairly interesting experience that i would do again if i could, like eating a pizza-burrito.
Unfortunately, since we made mistakes along the way, we didn't end up with a gel or results.