Week 3

Last week the supervisor of my lab actually decided for me to work on a different project that still connects with the previous project I was working on. Previously I was supposed to express and eventually purify the telomerase RNA binding domain(TRBD). In order to purify any protein, the protein has to have a specific tag that would help select for that only that protein. In this case, the TRBD protein was tagged with a maltose binding protein. However after purification, the maltose binding protein must be separated from the TRBD protein. In order to do this a specific protease is used. In this case a TEV protease encoded by a tobacco etch virus cleaves at specific site to separate the maltose binding protein from TRBD. This digestion process usually takes about a day. This image I got from https://www.neb.com/applications/protein-expression-and-purification/coupled-protein-expression-and-purification/maltose-binding-protein-expression may better describe what I am talking about.

                        
The TEV protease binds to and cleaves off a specific amino acid sequence located between TRBD and the maltose binding protein. The supervisor in my lab recently read about a single amino acid change in this sequence may allow the TEV protease to cleave off the sequence faster. This would be more efficient for purifying the TRBD protein because the digestion process may happen in less than two hours vs. an entire day. My project right now is to show how efficient the single amino acid change in the TEV cleavage sequence would be for purifying TRBD because the lab has never tested it before.
Because of this sudden project change the only hands on thing I actually did was make a buffer for dispensing the pellets containing TRBD as part of the purification for TRBD. The rest of the time I attended lab meetings where each person talked about their progress with their projects and when the supervisor tried to explain this project to me.
This week I will be starting the new project and in the next post I will talk more about it in detail. Like some other people, I may also have to change the title of my presentation.

Week 2

Last week I expressed the Telomerase RNA binding domain (TRBD) of sea urchin in bacterial cells.

By expression, I mean this gene has not yet transcribed and translated to become protein. In order for the TRBD gene to transcribe, the reagent (IPTG) must be present. IPTG functions similar to allolactose. It binds to the lac repressor on the operator (sequence in promotor) in bacteria in an allosteric manner (meaning IPTG binds to the lac repressor in a spot other than the active site) Through this binding, IPTG is able to change the shape of the repressor, disabling the lac repressor from binding to a sequence in the promotor. Once this happens, RNA polymerase is able to bind to the promotor and transcribe the TRBD gene into mRNA which can then translate into protein.

Previously my mentor had transformed E.coli bacterial cells with a plasmid containing the TRBD gene. In other words, there was already a stock solution of bacterial cells which contained a plasmid containing the TRBD gene. I had to isolate a colony of bacterial cells and eventually grow them up in a 1L culture before inducing protein expression with IPTG.

The first day I streaked the stock solution of bacterial cells onto a carbenicillin plate. Carbenicillin is an antibiotic used as a means of selecting only bacterial colonies resistant to this antibiotic. The cells were then incubated and allowed to proliferate overnight at 37 degrees celsius.

The next day I saw colonies of bacteria on the plate and isolated a single colony of bacteria through what is known as a starter culture. To do this first pipetted 10 microliters of a stock of carbenicillin in a glass culture tube. Once again I only wanted to grow up bacteria resistant to this antibiotic. I then added 10 ml of lysogeny broth to each of my culture tubes. Lysogeny broth is a liquid media rich with nutrients allowing bacteria to proliferate quickly. I then flamed forceps and picked a colony of bacteria with a sterile toothpick and placed that in the culture tube. I then allowed bacteria to grow in a shaking incubator at 37 degrees celsius overnight.
The same day I prepared one liter of the mixture of lysogeny broth for preparation to grow bacteria from the 10 ml culture to the 1L culture the next day. The reason why I first had to grow up bacteria in a 10 ml culture is because if something went wrong with the bacterial growth initially, it only ruined 10 ml of culture not a whole liter. You can tell that there was something wrong with the bacterial growth in 10 ml because the solution starts to turn more clear. In order to prepare the 1L culture, I mixed 25g of lysogeny broth mix with 1L of water. I then had to take this mixture to an autoclave machine which is a large pressure chamber that would sterilize the lysogeny broth media in 20 minutes.

The next morning, I added one ml of carbenicillin to the larger 1L culture along with 10 ml of 20% glucose. I then removed the initial 10 ml starter cultures from the shaking incubator and added it to the 1L media. After mixing the 1L media, I pipetted an aliquot of this media into a cuvette which I put in a spectrophotometer to obtain the initial optical density of the cell culture which is proportional to the cell density. I then placed this 1L media in a shaking incubator at 37 degrees celsius and periodically recorded the optical density till the bacterial culture solution reached a desired optical density. This desired optical density is usually approximately double the amount of the initial value.

After incubation, I could finally begin the protein expression. I added .05g of IPTG to the 1L bacterial culture solution and then placed the bacterial culture solution in a 25 degrees celsius shaking incubator. Protein expression was then carried out for four hours.

In the meantime, my mentor had me extract DNA from 10 PCR samples and then extract DNA from agarose gel samples. This was not specific to my project, it was done for practicing DNA extraction. I basically added various wash buffers and lysis buffers to the DNA solutions and finally measured the concentration of the DNA in each sample. The process was very similar to the mini prep from last week, however the term miniprep is used to talk about isolating specifically plasmid DNA in preparation for further usage of the plasmid.

After four hours, protein should have been expressed in the bacterial cultures. I then poured the 1L media into two 500 ml culture tubes which I had to centrifuge. Centrifugation would allow the bacterial cells with protein to settle at the bottom of the 500 ml tubes which would be called the pellet. Because the volume is so large, I used an ultracentrifuge which is capable of generating very high acceleration speeds; I centrifuged the cells at 5000 RCF (Relative Centrifugal Force measured by force generated multiplied by gravity) After centrifugation, I removed the media sitting on top and froze the pellets in -80 degrees celsius.

This week I will be purifying the protein in the pellets.  

  

Week 1

The first week I was given a couple articles to read through that explained more about the specific project I would be working on. I learned that the enzyme telomerase has four domains: the N-terminus domain (TEN), telomerase RNA binding domain (TRBD), the reverse transcriptase domain (RT), and the C terminus extension domain (CTE). My project would be ascertaining how the RNA component in telomerase is binding to TRBD. In order to this, I would be isolating the TRBD protein from telomerase and expressing it in bacterial cells. I would then be purifying the TRBD protein in an attempt to determine a crystal configuration of TRBD. 

In order to begin my project, I first had to learn about the techniques used to express proteins. I learned how to prepare protein and dna gel stock solutions and how to use an SDS Page instrument to isolate protein based on size. I also learned how to use a Kodak imager to visualize the size of protein I have. 

After observing others in the lab, the first task my mentor had me do by myself was a 'mini-prep'- a technique to isolate DNA from all other protein in the given sample. The mini prep I did was not specific to my project, I did it to practice the techniques I would be using for my project. My mentor needed only plasmid DNA in his sample so he could use that plasmid DNA as a vector or a means of inserting in a foreign DNA fragment for cloning a specific gene. In order to do this, I followed a protocol my lab had laid out which told me to add specific buffer reagents to first wash the the cells in the sample several times, break open the cells, and add neutralization buffer to keep the DNA inside the cells intact. 

After the mini prep, I used a nano drop to measure the concentration of DNA to protein. I first blanked the instrument with 1 microliter of water, then inserted one microliter of my sample to obtain an absorbance reading of my sample which I could use to calculate the concentration. Because I was supposed to be isolating DNA, the concentration of DNA was expected to be much higher than concentration of protein. Because the aromatic rings in DNA absorb light at 260 nanometers and the amino acids in protein absorb light at 280 nanometers, my sample should have absorbed significantly greater amount of light at 260nm than 280nm. 

 

This was the absorbance vs. wavelength graph of my two samples, and in this you can see that the mini prep I conducted was successful because there is a significantly higher absorbance at the wavelength DNA absorbs than the wavelength of light at which protein absorbs. However, it was unclear why one of my samples had a higher absorbance at 260 nm than the other sample because both the samples were supposed to be the same.

After obtaining these results, my mentor had me run the DNA sample on a gel so we can detect the size of DNA we had. Below are the band sizes of DNA I was able to visualize through a Kodak imager. The first column depicts the ladder I inserted in the first well of the gel which is supposed to be a marker or a reference to compare the size of my samples to. The two columns to the right of the ladder show the band sizes of my two samples. It was unusual that the Kodak imager displayed two bands instead of just one band and the sizes of the DNA samples seemed a little off. My mentor said that we would need to perform further experiments next week on this sample to see what went wrong and why the imager displayed two bands instead of one.   

Background information

Because DNA replicates in a 5’ to 3’ direction, the lagging strand of DNA running in a 3’ to 5’ direction tends to cut short as RNA primers are used to backstitch DNA. The ribonucleic protein, telomerase is a reverse transcriptase that elongates the lagging strand of DNA by adding repeating sequences of the polynucleotide “TTAGGG” to the 3’ ends of chromosomes called telomeres. Telomerase is beneficial for both normal and cancerous cells because it maintains cell sustainability and keeps cells from deteriorating. However normal cells tend to have small amounts of telomerase leading to shortening of telomere sequences and tissue degeneration; while tumor cells have been able to replicate indefinitely because they have been able to reactivate telomerase. The mechanisms allowing telomerase to do this remains unknown, and it is therefore important to study more about the telomerase reaction cycle and its RNA/DNA duplex. My project will be focusing on the RNA protein interactions in telomerase between the telomerase reverse transcriptase protein subunit and the telomerase RNA subunit.

Snippet about my project

The ribonucleoprotien, telomerase is a reverse transcriptase currently in research because of its potential use as an anti-aging as well as anti-cancer therapy. Although the structure of telomerase remains ambiguous, it was recently discovered that telomerase has its own RNA component. The interaction between the RNA sub unit and the protein subunit that it binds to in telomerase remains unknown. For my senior research project, I will be expressing the two sub units of telomerase in bacterial cells in an attempt to ascertain more about the structure and function of this complex enzyme.