I previously talked about how I expressed the TEV G>S cleavage site in bacterial cells. Not much happened last week, I was mostly focusing on working on my presentation. I scanned the SDS Page gel image I had that confirmed the TEV cleavage site was successfully expressed as protein. Then my mentor had to purify the protein because he said it was very complicated and needed everything to work well the first time. This week and next week I am running Bradford assays with the purified protein to determine the concentration of protein I have. The Bradford assay involves the use of Coomassie Brilliant Blue dye binding to specific protein of interest in order to determine the absorbance of that protein. Coomassie Brilliant Blue dye has three variant forms: red under acidic conditions, blue under basic conditions, and green under neutral conditions. When the dye binds to the protein of interest, it changes color from red to blue. The darker blue the dye is, the higher concentration of protein is bound to the dye. A spectrophotometer shines UV light through sample and detects the blue protein dye form at 595 nanometers of wavelength. Specific volumes of Coomassie Brilliant Blue dye will be added to known concentrations of the protein Bovine Serum Albumin and the absorbance of each variant solution will be detected with a spectrophotometer. A standard curve of absorbance vs. concentration will be generated with this to determine the unknown concentration of the protein I am testing.
Wednesday, April 20, 2016
Sunday, April 10, 2016
Week 7/8
Two weeks ago I attempted to express the cloned TEV cleavage site as a protein, however my results were not successful so I have been performing the same procedure last week and continuing on to this week as well. I previously discussed how I streaked a Carbenincillin plate and stored it for expression. I inoculated or isolated four colonies from this plate containing the TEV plasmid with the serine amino acid and grew it up overnight it 5 ml of bacterial culture tubes. I also isolated one colony from a previous glycerol stock containing the original TEV plasmid with the glycine amino acid and grew that up in 5 ml of bacterial culture overnight as well.
I then made SDS Page gels which I would use to later see whether my protein was actually expressed. (I was expressing the protein the next day, but making the gels in advance. The TEV plasmid would only be expressed as protein once induced with IPTG which I will talk about a bit later) SDS Page stands for sodium dodecyl sulfate polyacrylamide gel electrophoresis. This technique is used to separate proteins using two different buffer solutions. Sodium dodecyl sulfate is a detergent used to denature or unfold the protein from its tertiary state. This detergent also gives protein a negative charge giving proteins the desire to move towards the positive charge pole in the instrument also know as the cathode. Polyacrylamide forms the shape of the gel giving it its soft texture.
After growing up 5 ml cultures of the original TEV plasmid and the TEV plasmid with the changed amino acid, dispensed an aliquot of each bacterial culture and dispensed it in fresh 2 ml LB media and allowed the cultures to grow even longer. After an hour I checked the absorbance of each bacterial culture using a spectrophotometer. An absorbance reading between 0.3 and 0.4 means that the bacteria is now growing exponentially so its best to induce with IPTG at that specific time.
Once the bacterial cultures were growing exponentially, I dispensed an aliquot of each sample in SDS dye to denature the proteins. I labeled these sample tubes as uninduced samples because they were not induced with IPTG and therefore should not be expressed as protein. To the rest of the bacterial culture tubes, I added a calculated amount of IPTG. As I mentioned previously IPTG stands for Isopropyl β-D-1-thiogalactopyranoside and binds to the repressor which is bound to the promotor in lac operons. As IPTG binds to the repressor, the repressor changes shape and disassociates with the promotor allowing the gene to transcribe and translate to form protein. Expression for my specific protein takes about four hours. After inducing each bacterial culture with IPTG, I placed the culture samples in a shaking incubator at room temperature. After four hours I once again checked the absorbance of each sample and recorded the values. I then dispensed an aliquot of each culture sample in SDS dye and labeled these samples as the induced samples.
The next day I loaded equal and proportional amounts of both the induced and uninduced samples on the SDS Page gel. Once the proteins had been separated through the SDS Page gel, I stained the gel in Instant Blue dye so that I could visualize the bands of each protein sample. I then destained the gel in water because so that the dye would only remain on the protein bands and not the entire gel. After visualizing the gel on an imager, it was unclear to tell for sure whether the samples induced with IPTG actually expressed protein. It turns out that the IPTG solution I had used was a very old solution in the lab so last week I had to the same process all over again with a new IPTG solution. Last Friday I finished running the gel again and this Monday I will see if this time the protein expression was successful.
I then made SDS Page gels which I would use to later see whether my protein was actually expressed. (I was expressing the protein the next day, but making the gels in advance. The TEV plasmid would only be expressed as protein once induced with IPTG which I will talk about a bit later) SDS Page stands for sodium dodecyl sulfate polyacrylamide gel electrophoresis. This technique is used to separate proteins using two different buffer solutions. Sodium dodecyl sulfate is a detergent used to denature or unfold the protein from its tertiary state. This detergent also gives protein a negative charge giving proteins the desire to move towards the positive charge pole in the instrument also know as the cathode. Polyacrylamide forms the shape of the gel giving it its soft texture.
After growing up 5 ml cultures of the original TEV plasmid and the TEV plasmid with the changed amino acid, dispensed an aliquot of each bacterial culture and dispensed it in fresh 2 ml LB media and allowed the cultures to grow even longer. After an hour I checked the absorbance of each bacterial culture using a spectrophotometer. An absorbance reading between 0.3 and 0.4 means that the bacteria is now growing exponentially so its best to induce with IPTG at that specific time.
Once the bacterial cultures were growing exponentially, I dispensed an aliquot of each sample in SDS dye to denature the proteins. I labeled these sample tubes as uninduced samples because they were not induced with IPTG and therefore should not be expressed as protein. To the rest of the bacterial culture tubes, I added a calculated amount of IPTG. As I mentioned previously IPTG stands for Isopropyl β-D-1-thiogalactopyranoside and binds to the repressor which is bound to the promotor in lac operons. As IPTG binds to the repressor, the repressor changes shape and disassociates with the promotor allowing the gene to transcribe and translate to form protein. Expression for my specific protein takes about four hours. After inducing each bacterial culture with IPTG, I placed the culture samples in a shaking incubator at room temperature. After four hours I once again checked the absorbance of each sample and recorded the values. I then dispensed an aliquot of each culture sample in SDS dye and labeled these samples as the induced samples.
The next day I loaded equal and proportional amounts of both the induced and uninduced samples on the SDS Page gel. Once the proteins had been separated through the SDS Page gel, I stained the gel in Instant Blue dye so that I could visualize the bands of each protein sample. I then destained the gel in water because so that the dye would only remain on the protein bands and not the entire gel. After visualizing the gel on an imager, it was unclear to tell for sure whether the samples induced with IPTG actually expressed protein. It turns out that the IPTG solution I had used was a very old solution in the lab so last week I had to the same process all over again with a new IPTG solution. Last Friday I finished running the gel again and this Monday I will see if this time the protein expression was successful.
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