RFP lab
Purpose:
To make RFP in bacteria from Jelly fish, and learn about the genetic engineering steps.
Materials and Procedure:
2a- materials and procedure in Amgen manual part 2a
4a-materials and procedure in Amgen manual part 4a
5a-materials and procedure in Amgen manual part 5a
6-materials and procedure in Amgen manual part 6
Experimental overview:
Part 2a: Verification of plasmid by restriction digest
-Cut a plasmid with BanHI + HindIII to cut out RFP-ara gene from the bacteria plasmid
Part 4a: Verification of plasmid digest by electrophoresis.
Part 5a: Transformation of bacteria with recombinant plasmid
-Make bacteria into recombinant plasmid with restrictions
Part 6: Portification of RFP using chromatography.
Data:
Part 2a: 1.ori-origin of replication, GoI-gene of interest, Amp-R-selective marker, Ara-C-binds to the promoter so we can get transcription of our GoI. 2. restriction enzymes in nature are defense mechanisms that cut DNA from other bacteria. 3. Bacteria retains an antibiotic resistance because otherwise they would die off. In the medical field they have to create a whole new class of antibiotics. 4. Bacteria have DNA, RNA polymerase, and ribosomes (central dogma) just like humans. We can replace DNA with other DNA that can change a specific trait.
Part 4a:Since the recombinant plasmid has the desired trait you want to make sure that it really has the desired trait. 2. The gel results compared different to our comparisons because the DNA ladder was not visible due to a malfunction somewhere. 3. No I don't believe so, besides the opposite of not showing up. 4. Well without the DNA ladder we can't conclude this question as for evidence because we cannot estimate the measures. 5. No just one band. 6. RFP to R+ and AMP R to R-.
Part 5a: My predictions were correct but we did miss one of our pipeting instances where we crossed the middle line. 2. None. 3. They did not appear. 4. because not a lot is used as desirable, the more the better. 5. RFP gives the red flourescent glow to our proteins. 6. bacteria can make human protein because they have like organelles and cell processes.
Part 6: 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:
Unfortunately our lab didnt go exactly as planned. In the end of this lab, my group and i 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:
I worked with Logan and Yazeed on this project, and they were really good teammates. We got a lot done and we got everything done on time. we never fought as a group or had any complaints about each other. the only thing we had trouble with was pipetting in one of the slots, it made our results a little unclear.
Picture:
To make RFP in bacteria from Jelly fish, and learn about the genetic engineering steps.
Materials and Procedure:
2a- materials and procedure in Amgen manual part 2a
4a-materials and procedure in Amgen manual part 4a
5a-materials and procedure in Amgen manual part 5a
6-materials and procedure in Amgen manual part 6
Experimental overview:
Part 2a: Verification of plasmid by restriction digest
-Cut a plasmid with BanHI + HindIII to cut out RFP-ara gene from the bacteria plasmid
Part 4a: Verification of plasmid digest by electrophoresis.
Part 5a: Transformation of bacteria with recombinant plasmid
-Make bacteria into recombinant plasmid with restrictions
Part 6: Portification of RFP using chromatography.
Data:
Part 2a: 1.ori-origin of replication, GoI-gene of interest, Amp-R-selective marker, Ara-C-binds to the promoter so we can get transcription of our GoI. 2. restriction enzymes in nature are defense mechanisms that cut DNA from other bacteria. 3. Bacteria retains an antibiotic resistance because otherwise they would die off. In the medical field they have to create a whole new class of antibiotics. 4. Bacteria have DNA, RNA polymerase, and ribosomes (central dogma) just like humans. We can replace DNA with other DNA that can change a specific trait.
Part 4a:Since the recombinant plasmid has the desired trait you want to make sure that it really has the desired trait. 2. The gel results compared different to our comparisons because the DNA ladder was not visible due to a malfunction somewhere. 3. No I don't believe so, besides the opposite of not showing up. 4. Well without the DNA ladder we can't conclude this question as for evidence because we cannot estimate the measures. 5. No just one band. 6. RFP to R+ and AMP R to R-.
Part 5a: My predictions were correct but we did miss one of our pipeting instances where we crossed the middle line. 2. None. 3. They did not appear. 4. because not a lot is used as desirable, the more the better. 5. RFP gives the red flourescent glow to our proteins. 6. bacteria can make human protein because they have like organelles and cell processes.
Part 6: 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:
Unfortunately our lab didnt go exactly as planned. In the end of this lab, my group and i 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:
I worked with Logan and Yazeed on this project, and they were really good teammates. We got a lot done and we got everything done on time. we never fought as a group or had any complaints about each other. the only thing we had trouble with was pipetting in one of the slots, it made our results a little unclear.
Picture: