Week 6

Last week I finished cloning a plasmid construct containing the changed amino acid in the TEV cleavage site. To confirm whether the cloning was successful I picked four random colonies from the plate with the plasmid of interest and allowed them to grow overnight in lysogeny broth media. I then isolated specifically the plasmid DNA by performing mini preps for each plasmid and then used a nanodrop to obtain the concentration of plasmid DNA I had. I then took an aliquot of the purified DNA plasmid and diluted it with a specific primer (a sequence of DNA molecules that base pair with the DNA sequences in my plasmid, allowing DNA polymerase to extend the plasmid DNA sequence I have) I then submitted this mixture to a separate sequencing lab. What the sequencing lab basically did was amplify the DNA sequence I gave them in order to determine the exact sequence of the DNA I gave them. Basically how this works is through addition of dideoxynucleotides (nucleotides which lack oxygen or a free hydroxyl group) which have a fluorescent tag attached to them which allows us to visually see the DNA sequence through a computerized system. After the sequencing lab emailed back the DNA sequence I had, I was able to compare the amino acid sequence in the DNA samples I submitted with what the DNA sequence was supposed to look like. I had submitted four samples; two of the samples I submitted had the exact amino acid sequence as expected whereas the other two had errors. Because I was able to receive two successful clones of my plasmid, I could use one of them to express as a protein. I took an aliquot of the purified plasmid sample with positive cloning results and transformed that plasmid into two different bacterial strains and streaked these strains on two different plates. One bacterial strain containing my plasmid will be kept in storage in a -80 degrees Celsius temperature. The other bacterial strain containing my plasmid will be used to express my plasmid DNA as a protein. After expressing the DNA, I will purify the protein to ultimately run trials and see the efficiency at which the TEV protease cleaves off the TEV cleavage site with the change in amino acid.