Aileen San
BIO 210L – Section 4
A Prokaryotic System for Transformation, Expression, and Purification of Eukaryotic Proteins
Introduction: The bacteria E.coli is found in many places such as in animal intestines and the environment. These bacteria have a simple structure and are quick to reproduce (Centers for Disease Control and Prevention, 2015). Because of this, scientists know a lot about E. coli. In this experiment, the E. coli will be exposed to a pGLO plasmid; each plasmid has an Ori, pBAD, araC, bla, and GFP. The Ori, origin of replication, is the site of DNA replication in the plasmid. On these DNA strands is the pBAD promoter, a sequence of DNA used to start the transcription of the GFP (green fluorescent protein), which only …show more content…
This is calculated by multiplying the concentration of the pGLO stock solution to the amount used in µL. The next step was to determine how much DNA was actually plated. In order to calculate this, the amount of additional liquid added needs to be factored in; we added together the total amount of transformation solution used to the amount of LB nutrient broth and plasmid DNA stock solution in the ependorf tubes. Using this information, the amount of plasmid exposed to E. coli cultures was divided by the amount of extra liquid added in to find how much DNA actually ended up on the plate. Because transformation efficiency is usually expressed in terms of number of transformants per microgram of DNA, the number of colonies on the +pGLO/LB/amp/ara and +pGLO/LB/amp plates were counted then divided by the amount of plasmid DNA plated.
Induction of GFP …show more content…
Using a new pipette tube, 1 mL of the “W” liquid culture was transferred into another microfuge tube. The two tubes were then centrifuged for 5 minutes at maximum rpm. Almost all the supernatant from each tube was taken out and discarded back into the culture tubes. To each of the pellets in the microfuge tubes, 125 µL of TE buffer were added. With the pipette tip, the pellets were chopped up and mixed by pipetting up and down to resuspend the pellets in the tubes. The two suspensions were then combined into one tube and vortexed vigorously for about 30 seconds. With a P200 micropipette, 60 µL of lysozyme was added into the suspension to break the cell walls of the bacteria. Every five minutes during a 15-minute incubation period in 30˚C water, the contents of the tube were mixed with a pipette. After incubation, the bacteria were placed in the freezer for 15 minutes at -20˚C.
After removing the tube from the freezer, it was left to thaw then vortexed for 30 seconds. The tube was then placed into a centrifuge for 10 minutes to separate the ruptured bacterial cell walls. In a microfuge tube, 250 µL of the supernatant is transferred over and left to equilibrate for 5 minutes. When the supernatant was finished equilibrating, 500 µL of the supernatant was slowly dialed in to a protein purification
BIO 210L – Section 4
A Prokaryotic System for Transformation, Expression, and Purification of Eukaryotic Proteins
Introduction: The bacteria E.coli is found in many places such as in animal intestines and the environment. These bacteria have a simple structure and are quick to reproduce (Centers for Disease Control and Prevention, 2015). Because of this, scientists know a lot about E. coli. In this experiment, the E. coli will be exposed to a pGLO plasmid; each plasmid has an Ori, pBAD, araC, bla, and GFP. The Ori, origin of replication, is the site of DNA replication in the plasmid. On these DNA strands is the pBAD promoter, a sequence of DNA used to start the transcription of the GFP (green fluorescent protein), which only …show more content…
This is calculated by multiplying the concentration of the pGLO stock solution to the amount used in µL. The next step was to determine how much DNA was actually plated. In order to calculate this, the amount of additional liquid added needs to be factored in; we added together the total amount of transformation solution used to the amount of LB nutrient broth and plasmid DNA stock solution in the ependorf tubes. Using this information, the amount of plasmid exposed to E. coli cultures was divided by the amount of extra liquid added in to find how much DNA actually ended up on the plate. Because transformation efficiency is usually expressed in terms of number of transformants per microgram of DNA, the number of colonies on the +pGLO/LB/amp/ara and +pGLO/LB/amp plates were counted then divided by the amount of plasmid DNA plated.
Induction of GFP …show more content…
Using a new pipette tube, 1 mL of the “W” liquid culture was transferred into another microfuge tube. The two tubes were then centrifuged for 5 minutes at maximum rpm. Almost all the supernatant from each tube was taken out and discarded back into the culture tubes. To each of the pellets in the microfuge tubes, 125 µL of TE buffer were added. With the pipette tip, the pellets were chopped up and mixed by pipetting up and down to resuspend the pellets in the tubes. The two suspensions were then combined into one tube and vortexed vigorously for about 30 seconds. With a P200 micropipette, 60 µL of lysozyme was added into the suspension to break the cell walls of the bacteria. Every five minutes during a 15-minute incubation period in 30˚C water, the contents of the tube were mixed with a pipette. After incubation, the bacteria were placed in the freezer for 15 minutes at -20˚C.
After removing the tube from the freezer, it was left to thaw then vortexed for 30 seconds. The tube was then placed into a centrifuge for 10 minutes to separate the ruptured bacterial cell walls. In a microfuge tube, 250 µL of the supernatant is transferred over and left to equilibrate for 5 minutes. When the supernatant was finished equilibrating, 500 µL of the supernatant was slowly dialed in to a protein purification