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.

Week 5- Cloning Complete!

In my previous post I talked about how I cut the plasmid vector backbone and my PCR product with the same restriction enzymes. Last week, I then run the plasmid vector on an agarose gel to separate my vector backbone which was approximately 7,000 base pairs in size from the piece I did not want which was approximately 78 base pairs in size. Because of the large size difference, it was very easy to see the position my vector backbone was at and I extracted out the vector backbone by actually cutting it out with a blade. I then used the Gel extraction/PCR cleanup protocol I briefly described in previous posts to purify the plasmid DNA from extraneous salts and whatnot. At the same time I also purified my digested PCR product. After this was completed, I ligated the PCR product with the change in amino acid to the plasmid vector backbone.
 I then used electroporation to introduce the ligated DNA molecule containing the amino acid of interest into bacterial cells that can proliferate and make clones of my plasmid. Electroporators are basically that can generate an electric field in order to make bacterial cells permeable to the plasmid I am trying to insert into it. In this way I was able to transform bacteria so that it could take up the ligated DNA plasmid I had. I then heated the mixture containing the transformed bacterial cells and streaked these cells on a plate. I let colonies of bacterial cells to grow overnight in a 37 degrees incubator.
At the same time, I also used electroporation to introduce solely the plasmid vector backbone I had that did not contain my insert into bacterial cells. This was done to serve as a negative control. I streaked out these bacterial cells on a different plate to see whether for some reason I would see bacterial colonies grow. If they did, it would mean, I did something wrong and the plasmid ligated to itself instead of my insert. The plasmid was not supposed to ligate to itself because it was cut with two different restriction enzymes and making the ends of my plasmid incompatible to base pair with one another.
The following image shows the bacterial colonies I saw the next day.

Each small white circular dot is a bacterial colony. The plate on the left had the plasmid vector without the insert whereas the plate on the left had the ligated plasmid and PCR insert. There was one bacterial colony on the left plate, and over 30 bacterial colonies on the right plate. My mentor had said that 30:1 is a pretty high efficiency so basically the cloning seems to be successful.
This week however, I am screening for colonies, meaning I am picking 4 random colonies from the plate on the right to confirm that these colonies actually have the insert I am studying with the amino acid change. I will discuss more about the screening process in the next post.

Week 4

Previously I discussed how I am now changing an amino acid in the TEV cleavage site. (TEV stands for tobacco etch virus; it's the virus encoding this specific sequence)  I am specifically changing the amino acid glycine to serine so that the TEV protease can cleave off the sequence more efficiently. In order to do this I had to amplify and isolate a plasmid containing the TEV cleavage site with the glycine amino acid; this would serve as the backbone of a recombinant molecule I am trying to create. I would then cut out the TEV cleavage site with the glycine amino acid and replace that with a similar sequence with the exception of the serine amino acid change.
Last week I first streaked a bacterial strain on a plate containing the plasmid backbone I needed for the cloning procedure. (This is the plasmid with the glycine amino acid in the TEV cleavage site)I allowed the bacterial colonies to grow overnight at 37 degrees Celsius. The next day I isolated a single colony and allowed it to grow up in LB media overnight. I then performed a mini prep (the technique I talked about in my previous posts) to isolate specifically my plasmid of interest.
At the same time I also performed PCR (Polymerase Chain Reaction; a technique used to amplify DNA) on the TEV cleavage site with the serine amino acid in place of the glycine. To confirm that the PCR worked, I ran the PCR samples on a gel to visualize the DNA bands. My PCR did work because in the gel image, the negative controls I had did not show while my samples did. I then performed a PCR cleanup to once again make sure I only have the DNA needed and not any other extraneous things. I obtained the concentration of my PCR samples with a nano drop.
After amplifying and isolating both the plasmid backbone and the PCR samples, I used the same restriction enzymes to digest or cut both the plasmid and the PCR samples. (restriction enzymes can be thought of as scissors used to cut DNA) By using the same restriction enzymes for the plasmid and PCR samples, I was able to create the same type of cuts which could ligate or bind to one another.
Today I ran the digested plasmid vector on a gel to separate basically the large backbone which I want from the small TEV site with a glycine amino acid which I don't want. I then cut out the gel piece with the plasmid and tomorrow I will be purifying it. After purification I can then ligate (or "glue together") digested purified PCR product and digested purified backbone.