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.
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.
Excellent progress!
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