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17 thoughts on “P90 – Beck”
How would you utilized/edit Carl20 to make it work sepcifically for bacteria in humans? Would this involve gene editing, or would it be able to be used as a therpudic as is?
Hi, great question! Carl20 was isolated from Mycobacterium smegmatis, which you may recognize as a cousin (part of the same genus) as Mycobacterium tuberculosis. Research has shown that phage isolated from one species of bacteria are sometimes able to also infect a different species within the same genus. This has been shown many times from M. smeg to M. tuberculosis, so the hope is that Carl20 will be able to also. We would need to test this, of course. As for the second part of your question, phage therapy still has a lot of work to do. I answered why for Madison’s question below, but the short answer is, yes. Some gene editing will likely be needed, but not for the reasons you’re talking about. The main reason is that the body’s immune system is very good at attacking foreign objects, so scientists need to find a way to make it attack the tuberculosis or phage infected tuberculosis, but not the phage itself or the M. smeg lysogens being used as a transport mechanism. Please see my reply to Madision’s question for a little more detail on this. I also have some interesting sources there that follow with your question.
Could your phage be a mix of lysogenic and lytic phage since there are some clear plaques along with cloudy plaques? Or is this an indication that more than one type of phage is present?
Yes to your first question! The cloudy plaques mean there are Carl20s that underwent the lytic lifecycle and Carl20s that underwent the lysogenic lifecycle. Went a phage undergoes the lytic lifecycle, it always kills the bacteria, leading clear plaques. The phage particles burst out and go on to infect other nearby bacteria. When it undergoes the lysogenic lifecycle, the bacteria lives on (perhaps unhappily) with a small piece of Carl20 DNA inside of it, replicating the Carl20 DNA with the bacterial DNA when it divides. A phage is called temperate if it can undergo the lysogenic lifecycle. Here’s the thing though. That Carl20 lysogen continues to live and replicate, but eventually something will happen to the represser gene. Perhaps there’s a mutation or it’s smacked with some UV light. Either way, it breaks and it stops repressing the lytic genes. Now the lytic genes are transcribed and that Carl20 lysogen will now undergo the lytic lifecycle and burst and die, leading to clearer plaques. Now you may be asking why all the new Carl20 phage don’t immediately kill all of the other bacteria in the area and make a clear plaque. This is due to several reasons. One is that Carl20 is still a temperate phage, so even the new phage particles will still only infect but not kill the M. smeg. The other is that Carl20 lysogens are actually immune to infection by other Carl20s. A good way to think about it at first glance is that there is already Carl20 DNA at the bacterial chromosome’s infection site, so although other Carl20s will land on the lysogen and inject their DNA inside, their DNA won’t be able to integrate into the bacterial chromosome because there’s already something there! So to give the final answer to your questions, a temperate phage’s plaques are cloudy because there are some bacteria that underwent the lytic lifecycle and others that underwent the lysogenic lifecycle. To your second question, no. It’s just Carl20 in two different lifecycles!
Do you know why the phage is called Carl20? If so, could you explain why? I just find it interesting that the phage is named after an actual human name and not something more scientific.
I named it! I wanted it to be named Carl because I though the name was funny for a virus. It turns out someone else thought so too, as the name was already taken by a phage discovered at the University of Pittsburgh. Instead, it was named Carl20 because it was discovered in 2020.
Check out the phage database website: https://phagesdb.org/phages/
There are a lot of hilarious names that researchers chose!
In what ways could Carl20 be modified to be useful in phage therapy? As in, you mentioned lytic phages work better for phage therapy, but Carl20 is lysogenic; why do you think Carl20 could still be useful in phage therapy?
Great question! This is something I didn’t have time to get to in my presentation. You’re right that lytic phage works better for phage therapy because it’s the lytic phage that are actually able to consistently kill the bacteria, while temperate phage spend a lot of time lying dormant. Your question actually leads to some of the issues with phage therapy and why we still need to need to use antibiotics. For one thing, phage have a super high rate of clearance from the body. When they’re not infecting bacteria, they kind of just do nothing then get flushed out. An additional problem is that a large influx of phage into the body often produces an immune response, despite not actively causing harm to the body’s cells. Now back to your question. Say you’re using phage therapy to treat someone with Tuberculosis. A proposed way to deal with the above problems is to use the non-pathogenic M. smeg paired with a temperate phage to create lysogens that act as transport devices throughout the body. If the M. smeg can be engineered in a way that the immune system won’t immediately kill it, then these transport devices can move around the body, causing no harm (because it’s M. smeg and not M. tuberculosis). Now you can attack M. tuberculosis throughout the body. You can likely see several issues with this logic, but I think it’s a very promising idea, and a great way for Carl20 to get involved… we still need to see if it can infect M. tuberculosis. I guess we’ll see!
More information if you’re interested:
Phage therapy (requires free account): https://www.the-scientist.com/cover-story/viral-soldiers-34289
How to use phage therapy to fight Tuberculosis: https://www.bmbtrj.org/article.asp?issn=2588-9834%3Byear%3D2018%3Bvolume%3D2%3Bissue%3D1%3Bspage%3D9%3Bepage%3D15%3Baulast%3DGondil
Did you have an expected outcome? What was your hypothesis?
Not at the beginning, no. The purpose of this class was to find and classify a novel bacteriophage, so at the beginning of the semester, I was enriching soil samples with M. smeg in order to find a bacteriophage. When I finally found one, the plaques were large and cloudy. At that point the hypothesis was that the phage was temperate, but further classification needed to be done. After the restriction digest, the hypothesis was that Carl20 was of cluster A1.
What results were you expecting to see before you completed your experiments and how did these expected results compare to the actual results?
The purpose of this class was to find and classify a novel bacteriophage, so at the beginning of the semester, I was enriching soil samples with M. smeg. I was expecting to find plaques, indicating the presence of phage. When I finally found one, the plaques were large and cloudy. At that point the hypothesis was that the phage was temperate, but further classification needed to be done. After the restriction digest, the hypothesis was that Carl20 was of cluster A1. From there, I can look at the electron micrograph morphology and the plaque morphology and compare them to other A1 phage to find they look similar. At this point, however, it’s still a hypothesis. A PCR needs to be performed to confirm that Carl20 is of cluster A1.
Great job! You and Max accomplished so much this semester and you should be proud!
Thank you, Kylie, for explaining science to me throughout the semester!