Yes, this could be due to the differences in the time of infection. Newer plaques may be smaller in size because less bacteria have been lysed in that particular area. When we went onto purification rounds after the one pictured (we had several since we had a third person, Keala Gapin, working with us), the morphology was fairly consistent, but not exact. Still close enough in size and overall appearance to determine it was probably one type of phage!
Do you have any limitations in your experiment that lower the certainty of your findings such as ingrained inaccuracy of any techniques used in your lab?
The PCR and Restriction digest results have variability because they use enzymes to cut the DNA and that is used to estimate size and number of cuts seen in the DNA.
There are some limitations concerning the PCR and restriction digest experiments since the cuts made during the digests utilizing different enzymes are sometimes hard to count. Oftentimes, we undercount the cuts made during the restriction experiments since they can clump together and be faint in appearance, which can impact the recommended clusters later tested in the PCR experiments when the data is plugged into the phage enzyme tool. Amplification is also up for interpretation in the PCR, so making a final decision on the clustering of the phage needs to be done using sequencing and the PCR more so as a supplement.
We did a soil enrichment. Basically we put soil and a phage media into a flask and incubated it for 48 hours, then we drained the fluid and filtered the phage.
This process is soil enrichment- essentially, I took a soil sample from an area near the lab and added m. smeg and media to feed this bacteria into the sample. This soil sample was then incubated (giving the bacteria time to replicate so the phage would have something to infect). It was then filtered and plated and small plaques signaled that the sample of soil contained phage.
Any bacterial infection has the potential to be treated using phage therapy if a type of phage can be found that are able to infect it. The phage we isolated in particular have known host ranges that include m. smeg and m. tuberculosis, but could maybe infect other types of bacteria. That’s the cool thing about phage therapy- each new phage discovered offers new opportunities to treat different bacterial infections!
It is helpful to know the location for research purposes- if Wormels was pulled up in the data base and had the potential to be used for some more research, it would be helpful to know the type of conditions it was thriving in beforehand. Researchers could go back to the sample site and possibly isolate another Wormels or another type of phage that may be related, or very different, if need be.
The host range of a phage- meaning the bacteria it is able to infect- depends on the receptors on the bacterial cell and the mechanisms of the phage. If a phage can’t infect a certain strain of bacteria, this is probably because it doesn’t have the proper binding tools to attach to the cell and insert its DNA into the cell (since receptors are specific). Lytic phage are necessary for phage therapy as these are the ones that lyse the bacteria and can effectively treat the infection in an individual. Temperate phage kill bacteria much more slowly since they use the cell as a host, not to replicate. If a temperate phage has the potential to infect a bacterial cell and is needed for therapy, it can be altered to go into the lytic cycle so it can be used.
You are able to tell the life cycle of a bacteriophage by the appearance of the plaques they leave behind: temperate phage leave cloudy plaques and lytic phage leave clear plaques. Temperate phage, which are in the lysogenic life cycle, can slip into the lytic cycle if the repressor protein is prevented from binding since this protein is what prevents the phage from transitioning to the lytic cycle and lysing the cell. As mentioned in the comment above, lytic phage are what can be used in phage therapy since they rapidly lyse the bacteria cells and can get rid of the infection, so it is important we know the life cycle they enter so that they can be used for treatment. Temperate phage can be altered so they can become lytic and can effectively lyse cells if they are needed for a certain infection.
Why do you think that your plaques in figure 2 were of such different size and did that have any effect on your conclusions?
The different plaque sizes are likely from the time of phage infection. The earlier the infection the smaller the plaque.
Yes, this could be due to the differences in the time of infection. Newer plaques may be smaller in size because less bacteria have been lysed in that particular area. When we went onto purification rounds after the one pictured (we had several since we had a third person, Keala Gapin, working with us), the morphology was fairly consistent, but not exact. Still close enough in size and overall appearance to determine it was probably one type of phage!
Do you have any limitations in your experiment that lower the certainty of your findings such as ingrained inaccuracy of any techniques used in your lab?
The PCR and Restriction digest results have variability because they use enzymes to cut the DNA and that is used to estimate size and number of cuts seen in the DNA.
There are some limitations concerning the PCR and restriction digest experiments since the cuts made during the digests utilizing different enzymes are sometimes hard to count. Oftentimes, we undercount the cuts made during the restriction experiments since they can clump together and be faint in appearance, which can impact the recommended clusters later tested in the PCR experiments when the data is plugged into the phage enzyme tool. Amplification is also up for interpretation in the PCR, so making a final decision on the clustering of the phage needs to be done using sequencing and the PCR more so as a supplement.
What was the process of extracting your phage from the soil where you found it?
We did a soil enrichment. Basically we put soil and a phage media into a flask and incubated it for 48 hours, then we drained the fluid and filtered the phage.
This process is soil enrichment- essentially, I took a soil sample from an area near the lab and added m. smeg and media to feed this bacteria into the sample. This soil sample was then incubated (giving the bacteria time to replicate so the phage would have something to infect). It was then filtered and plated and small plaques signaled that the sample of soil contained phage.
What other bacteria besides M. tuberculosis are good potential candidates for phage therapy?
Any pathogenic bacteria is a good option for phage therapy. The reason M. tb was focused on is because tuberculosis it is a commonly known disease.
Any bacterial infection has the potential to be treated using phage therapy if a type of phage can be found that are able to infect it. The phage we isolated in particular have known host ranges that include m. smeg and m. tuberculosis, but could maybe infect other types of bacteria. That’s the cool thing about phage therapy- each new phage discovered offers new opportunities to treat different bacterial infections!
Why does the location of the where the microphage matter?
The location is important because it allows someone to replicate the experiment as closely as possible.
It is helpful to know the location for research purposes- if Wormels was pulled up in the data base and had the potential to be used for some more research, it would be helpful to know the type of conditions it was thriving in beforehand. Researchers could go back to the sample site and possibly isolate another Wormels or another type of phage that may be related, or very different, if need be.
Are there any specific characteristics of bacterium that make a certain species more suitable as a candidate for phage therapy relative to others?
Each bacterium has a phage that infects and lysis it meaning they could all be treated through phage therapy.
The host range of a phage- meaning the bacteria it is able to infect- depends on the receptors on the bacterial cell and the mechanisms of the phage. If a phage can’t infect a certain strain of bacteria, this is probably because it doesn’t have the proper binding tools to attach to the cell and insert its DNA into the cell (since receptors are specific). Lytic phage are necessary for phage therapy as these are the ones that lyse the bacteria and can effectively treat the infection in an individual. Temperate phage kill bacteria much more slowly since they use the cell as a host, not to replicate. If a temperate phage has the potential to infect a bacterial cell and is needed for therapy, it can be altered to go into the lytic cycle so it can be used.
Is there any way to know what life cycle the bacteriophages enter? Why would it be important in terms to your research?
You are able to tell the life cycle of a bacteriophage by the appearance of the plaques they leave behind: temperate phage leave cloudy plaques and lytic phage leave clear plaques. Temperate phage, which are in the lysogenic life cycle, can slip into the lytic cycle if the repressor protein is prevented from binding since this protein is what prevents the phage from transitioning to the lytic cycle and lysing the cell. As mentioned in the comment above, lytic phage are what can be used in phage therapy since they rapidly lyse the bacteria cells and can get rid of the infection, so it is important we know the life cycle they enter so that they can be used for treatment. Temperate phage can be altered so they can become lytic and can effectively lyse cells if they are needed for a certain infection.