Genetic mutations are the source of numerous hereditary diseases. This has been discovered by the comparison of two DNA sequences with bioformatic software. Two sequences are aligned and compared in order to locate mutations in the DNA (Module 4 Protocol,1). Escherichia coli aka E. coli was used as the test subject in this lab because it employs lac operons while transcribing mRNA. An operon is a unit of linked genes that regulates genes in charge of protein synthesis. A lac operon requires the presence of lactose to function. E. coli will only transcribe mRNA for specific enzymes when a certain sugar is detected in the surrounding environment. Lac operons are made up of a CAP binding site, -35/-10 sites, operator region, the start site
…show more content…
The nucleotide sequences for the wild type DNA and the mutant DNA, pLac/WT and pLac/m2, where aligned and compared to uncover any mutations. The locations of the CAP binding site, promoters, operator, and coding region needed to be highlighted and marked on the nucleotide sequences. The CAP binding site is located 105-116 on the nucleotide. Promoter -35 can be found from 140 to 145 on the sequence. The Promoter -10 is found from 164-169. The operator-binding site for the Lacl protein is located 172-192 on the sequences. The coding region for the ßGAL protein is positioned from 214-216. Mutations can occur as substitutions, deletions, or insertions. In the second part of week one the program TACG was used to detect restriction enzymes that cut the DNA sequences. Restriction enzyme maps are useful because it can show you a difference between the wild type DNA and the mutation DNA. Certain enzymes will cut the WT sequence and not the mutant or vice versa. The enzymes also may cut or not cut the wild type or the mutant multiple times. The restriction map shows the position of the restriction enzyme cut on the DNA sequence and what the size of the DNA fragment is. In week two the DNA sequences of the wild type and mutant were exposed to restriction enzymes and then agarose gel electrophoresis. The students prepared six tubes for the agarose gel. All of the pipetting was done with a P20 pipette. The controlled factors of this experiment were the amounts pipetted and the chosen restriction enzyme BsrG1. Tube A and Tube B were the negative controls, Tube A received 10ul of sterile water and 10ul of WT plasmid DNA. Tube B received 10ul or sterile water and 10ul of m2 Mutant plasmid DNA. Tube C and Tube D were the positive controls. Tube C and D both received 4ul of sterile water and reaction buffer. Tube C was also given 10ul of WT plasmid DNA and 2ul of the
The nucleotide sequences for the wild type DNA and the mutant DNA, pLac/WT and pLac/m2, where aligned and compared to uncover any mutations. The locations of the CAP binding site, promoters, operator, and coding region needed to be highlighted and marked on the nucleotide sequences. The CAP binding site is located 105-116 on the nucleotide. Promoter -35 can be found from 140 to 145 on the sequence. The Promoter -10 is found from 164-169. The operator-binding site for the Lacl protein is located 172-192 on the sequences. The coding region for the ßGAL protein is positioned from 214-216. Mutations can occur as substitutions, deletions, or insertions. In the second part of week one the program TACG was used to detect restriction enzymes that cut the DNA sequences. Restriction enzyme maps are useful because it can show you a difference between the wild type DNA and the mutation DNA. Certain enzymes will cut the WT sequence and not the mutant or vice versa. The enzymes also may cut or not cut the wild type or the mutant multiple times. The restriction map shows the position of the restriction enzyme cut on the DNA sequence and what the size of the DNA fragment is. In week two the DNA sequences of the wild type and mutant were exposed to restriction enzymes and then agarose gel electrophoresis. The students prepared six tubes for the agarose gel. All of the pipetting was done with a P20 pipette. The controlled factors of this experiment were the amounts pipetted and the chosen restriction enzyme BsrG1. Tube A and Tube B were the negative controls, Tube A received 10ul of sterile water and 10ul of WT plasmid DNA. Tube B received 10ul or sterile water and 10ul of m2 Mutant plasmid DNA. Tube C and Tube D were the positive controls. Tube C and D both received 4ul of sterile water and reaction buffer. Tube C was also given 10ul of WT plasmid DNA and 2ul of the