The effect of the BsrG1 restriction enzyme on Wild Type and Mutation 1 of E. coli DNA
Introduction:
What a lac operon is involved in the lactose metabolism of E. coli and it can only work when there is no glucose present to interfere with the lactose metabolism. (Khan Academy) E. coli chooses prefers glucose and other "better" sugars, so if any are present other than lactose the lac operon will not be expressed in the plasmid. (Khan Academy)
A plasmid is an "independent, circular, self-replication DNA molecule" that contrasts with the more complex helical shaped DNA of Eukaryotes. (Arizona State University) This type of DNA is often found in bacteria, specifically E. coli.
The lab group used E. coli as the test organism for this experiment, …show more content…
coli and a mutated plasmid. This lab group got mutation 1. The groups then used a program called Putty to view the bioinformatics program Muscle. Through Putty, each plasmid's DNA was sequenced to the base pair nucleotide letters. This enabled the groups to view the discrepancies between the genetic codes. Putty was opened on lab computers on the first day of this lab. Next, the group used Putty to input potential restriction enzymes to see where they would cut the DNA, and how long each of these fragments was. The restriction enzymes observed through the program were AseI, BsrGI, ClaI, EcoRV, HindIII, HpaI, NcoI, and PvuII. The lab group then chose to use BsrGI for the experimental restriction enzyme since they believed it would make the mutant DNA linear since it would cut the plasmid once while leaving the wild-type DNA uncut and supercoiled. This would make the wild-type DNA travel faster through the agarose gel in gel electrophoresis. They also chose a positive control group with the HindIII restriction enzyme that would cut both the mutant 1 and the wild type plasmid once and at the same position. Then they had a wild type and mutant 1 plasmids that had not been exposed to any restriction enzymes for a control …show more content…
The mutation 1 plasmid had a substitution mutation at the 173-base pair. The lab group used the bioinformatics program muscle to align the genetic codes of the two plasmids for comparison, which the group used a computer program called Putty to log in and view. The lab group assumed that lanes A, B, and E would move further down the hyperladder because they were uncut and supercoiled allowing them to travel further through the gel at a faster rate than those strands that were cut because of restriction enzymes. However, after further discussion between the lab group and the instructors, the lab group found that most plasmids had been nicked, uncoiling them but keeping them as a circular shape, making them much slower. So, the lab group decided to change their predictions that lanes A, B, and E would all be above the hyper ladder. The group predicted that lanes C, D, and F would all be at the 5,991-base pair mark, right below the 6kb on the hyperladder, because these plasmids were all the same length and had one clean cut making them a linear 5,991 base pair length DNA strand.
The group's hypothesis was supported. It is difficult to make out on the image for lane B because too little of the substance was placed into the gel electrophoresis well, however, all the lines ended in the approximate are that the lab group had
Introduction:
What a lac operon is involved in the lactose metabolism of E. coli and it can only work when there is no glucose present to interfere with the lactose metabolism. (Khan Academy) E. coli chooses prefers glucose and other "better" sugars, so if any are present other than lactose the lac operon will not be expressed in the plasmid. (Khan Academy)
A plasmid is an "independent, circular, self-replication DNA molecule" that contrasts with the more complex helical shaped DNA of Eukaryotes. (Arizona State University) This type of DNA is often found in bacteria, specifically E. coli.
The lab group used E. coli as the test organism for this experiment, …show more content…
coli and a mutated plasmid. This lab group got mutation 1. The groups then used a program called Putty to view the bioinformatics program Muscle. Through Putty, each plasmid's DNA was sequenced to the base pair nucleotide letters. This enabled the groups to view the discrepancies between the genetic codes. Putty was opened on lab computers on the first day of this lab. Next, the group used Putty to input potential restriction enzymes to see where they would cut the DNA, and how long each of these fragments was. The restriction enzymes observed through the program were AseI, BsrGI, ClaI, EcoRV, HindIII, HpaI, NcoI, and PvuII. The lab group then chose to use BsrGI for the experimental restriction enzyme since they believed it would make the mutant DNA linear since it would cut the plasmid once while leaving the wild-type DNA uncut and supercoiled. This would make the wild-type DNA travel faster through the agarose gel in gel electrophoresis. They also chose a positive control group with the HindIII restriction enzyme that would cut both the mutant 1 and the wild type plasmid once and at the same position. Then they had a wild type and mutant 1 plasmids that had not been exposed to any restriction enzymes for a control …show more content…
The mutation 1 plasmid had a substitution mutation at the 173-base pair. The lab group used the bioinformatics program muscle to align the genetic codes of the two plasmids for comparison, which the group used a computer program called Putty to log in and view. The lab group assumed that lanes A, B, and E would move further down the hyperladder because they were uncut and supercoiled allowing them to travel further through the gel at a faster rate than those strands that were cut because of restriction enzymes. However, after further discussion between the lab group and the instructors, the lab group found that most plasmids had been nicked, uncoiling them but keeping them as a circular shape, making them much slower. So, the lab group decided to change their predictions that lanes A, B, and E would all be above the hyper ladder. The group predicted that lanes C, D, and F would all be at the 5,991-base pair mark, right below the 6kb on the hyperladder, because these plasmids were all the same length and had one clean cut making them a linear 5,991 base pair length DNA strand.
The group's hypothesis was supported. It is difficult to make out on the image for lane B because too little of the substance was placed into the gel electrophoresis well, however, all the lines ended in the approximate are that the lab group had