Akshay Mody week 2 + 3
I am having a very formative experience in my lab thus far, and in weeks 2 + 3, I began to master the techniques that will help me be successful in my project.
Week 2 focused on once again mastering some of the techniques learned in week 1. Phages need bacteria as a host, so bacterial cultures are needed to be made constantly. In addition, I had not realized how important is to calculate the Colony Forming Units, of CFU. This is important because every bacterial strain is subject to different conditions and can vary in its ability to make colonies. We are working with the 4 strains of the shigella baceteria, and targeting this bacteria with a library of phages. We needed to use the streaked plates of the 4 strains, and put Bottom Layer LB and a respective colony from each strain into a flask and then put it in the shaking machine to make an overnight culture. However, the technique consuming most of our time in the lab has been the process of tittering the various phages by first amplifying the phage and then plating them. Because viruses are significantly smaller than bacteria, we made a serial dilution to the 10 to the minus 8 and combined this with the wild type shigella bacteria to see if plaques formed. If plaques formed, we calculated the PFU and made a conclusion about the specific phage receptors to the wild type bacterial strain. During the spotting of the phage, phage 5s did not plaque on either the OMP C- or the double knockout, leading us to conduct a tittering on all 4 strains using one dilution to the 10 -8.
Our research is focusing on 4 strains of the shigella bacterium. 1 wild type strain, 1 OMP A knockout, 1 OMP C knockout, and 1 double knockout. We are spotting and tittering several phage to see if any use the OMP A or OMP C as a receptor for infecting the bacterial strain. In addition, because Shigella is involved in creating colonies in the gut, we hope to potentially find a genetic tradeoff if we subjugate the bacterial strain to the phages because it will be forced to change its receptors. After week 3, we found that the six phages that we were working with for the most part spotted on all the bacterial strains. However, the phage 5s failed to spot on the OMP A or OMPC knockout, leading us to believe that the phage needs these specific proteins as receptors for the bacteria. We performed an efficiency of plating thereafter, but the results were inconclusive and we will perform one next week. In addition, we make more media and plates this week. Next week, we will begin tittering a new library of phages and see if we can find anything interesting about the phage receptors.
Week 2 focused on once again mastering some of the techniques learned in week 1. Phages need bacteria as a host, so bacterial cultures are needed to be made constantly. In addition, I had not realized how important is to calculate the Colony Forming Units, of CFU. This is important because every bacterial strain is subject to different conditions and can vary in its ability to make colonies. We are working with the 4 strains of the shigella baceteria, and targeting this bacteria with a library of phages. We needed to use the streaked plates of the 4 strains, and put Bottom Layer LB and a respective colony from each strain into a flask and then put it in the shaking machine to make an overnight culture. However, the technique consuming most of our time in the lab has been the process of tittering the various phages by first amplifying the phage and then plating them. Because viruses are significantly smaller than bacteria, we made a serial dilution to the 10 to the minus 8 and combined this with the wild type shigella bacteria to see if plaques formed. If plaques formed, we calculated the PFU and made a conclusion about the specific phage receptors to the wild type bacterial strain. During the spotting of the phage, phage 5s did not plaque on either the OMP C- or the double knockout, leading us to conduct a tittering on all 4 strains using one dilution to the 10 -8.
Our research is focusing on 4 strains of the shigella bacterium. 1 wild type strain, 1 OMP A knockout, 1 OMP C knockout, and 1 double knockout. We are spotting and tittering several phage to see if any use the OMP A or OMP C as a receptor for infecting the bacterial strain. In addition, because Shigella is involved in creating colonies in the gut, we hope to potentially find a genetic tradeoff if we subjugate the bacterial strain to the phages because it will be forced to change its receptors. After week 3, we found that the six phages that we were working with for the most part spotted on all the bacterial strains. However, the phage 5s failed to spot on the OMP A or OMPC knockout, leading us to believe that the phage needs these specific proteins as receptors for the bacteria. We performed an efficiency of plating thereafter, but the results were inconclusive and we will perform one next week. In addition, we make more media and plates this week. Next week, we will begin tittering a new library of phages and see if we can find anything interesting about the phage receptors.
Outside of the lab, I am training for the upcoming cross country season and exploring the beautiful campus of Yale.
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