Hi Mia! A ‘cluster’ is a classification for grouping species of phage with 50% or more overall genomic similarity (at the nucleotide-sequence level). There are also ‘sub-clusters’, which further divide phages in the same cluster into smaller groups which share at least 70% genomic similarity. The reasons we’d like to make a successful cluster prediction for our phage are twofold; first, it’s useful to have novel bacteriophages organized in the database so that future researchers can easily access species of phage with the genetic characteristics they’re most interested in (whether for therapeutic research or any other reason). Organizing by cluster, or genomic similarity, is one useful method of organization. Second, phages with similar genomes are often suited to attack similar kinds of bacteria. This can be useful for researchers looking to create phage ‘cocktails’ for therapy, which are combinations of multiple phages (similar or different) used to attack an infection all at once so that clearing of bacteria is maximized.
Hope this helps! 🙂
Hi Brian! I was thinking of which cluster or sub-cluster the phage might be assigned to. We want to complete tests using phage DNA which allow us to predict which other species our novel phage might be similar to, and eventually sequence the genome of our phage to confirm that prediction. A ‘cluster’ is a classification for grouping species of phage with 50% or more overall genomic similarity (at the nucleotide-sequence level). There are also ‘sub-clusters’, which further divide phages in the same cluster into smaller groups which share at least 70% genomic similarity. Once we have a cluster prediction for our phage based on the results of restriction digest and PCR experiments, and confirmation based on getting the phage genome sequenced, we will know which other species share at least 50%-70% genomic similarity with our phage and ultimately, ‘where the phage fits in the database’.
Thanks for the question Laura!
Serial dilutions were part of the protocol for producing a ‘High Titer Lysate’, or a highly concentrated solution of phage particles. The first step is to scrape phage from our streak purification plate (ensuring we have one new phage with unique and consistent morphology) into a tube of phage buffer. This creates a baseline concentration of phage, which we expect to be really strong and clear all the bacteria when plated onto M. smeg. We perform serial dilutions on that tube, resulting in 5 phage samples with decreasing concentration. When all 5 samples are plated, we expect to see a decreasing amount of plaques on each subsequent plate as the dilution level increases (and the phage concentration decreases). This is ideal because we need a ‘web-patterned’ plate (lots of overlapping plaques with bacterial lawn still visible in between), not a completely cleared plate, to produce our High Titer Lysate and eventually calculate the titer, or concentration of phage particles, in our original phage sample. In the end this is crucial information for archiving the phage and figuring out its infection pattern. Hope this clarifies some things!
Hi Lea! Great question, it definitely could be! M. smegmatis is often used in bacteriophage research as it is very similar to the bacteria M. tuberculosis, for example, which as we know is very pathogenic toward humans unlike M. smeg. This makes it useful to isolate phages which infect M. smeg, because phages can often infect species of bacteria which are very similar to one another genomically and morphotypically. Building a large database of M. smeg infecting phages could benefit research into phage therapies for tuberculosis infections in humans going forward. 🙂
What is a cluster and why is it important to test for it if you were to continue your phage research?
Hi Mia! A ‘cluster’ is a classification for grouping species of phage with 50% or more overall genomic similarity (at the nucleotide-sequence level). There are also ‘sub-clusters’, which further divide phages in the same cluster into smaller groups which share at least 70% genomic similarity. The reasons we’d like to make a successful cluster prediction for our phage are twofold; first, it’s useful to have novel bacteriophages organized in the database so that future researchers can easily access species of phage with the genetic characteristics they’re most interested in (whether for therapeutic research or any other reason). Organizing by cluster, or genomic similarity, is one useful method of organization. Second, phages with similar genomes are often suited to attack similar kinds of bacteria. This can be useful for researchers looking to create phage ‘cocktails’ for therapy, which are combinations of multiple phages (similar or different) used to attack an infection all at once so that clearing of bacteria is maximized.
Hope this helps! 🙂
What do you mean by, “Where the phage fits in the database.”
Hi Brian! I was thinking of which cluster or sub-cluster the phage might be assigned to. We want to complete tests using phage DNA which allow us to predict which other species our novel phage might be similar to, and eventually sequence the genome of our phage to confirm that prediction. A ‘cluster’ is a classification for grouping species of phage with 50% or more overall genomic similarity (at the nucleotide-sequence level). There are also ‘sub-clusters’, which further divide phages in the same cluster into smaller groups which share at least 70% genomic similarity. Once we have a cluster prediction for our phage based on the results of restriction digest and PCR experiments, and confirmation based on getting the phage genome sequenced, we will know which other species share at least 50%-70% genomic similarity with our phage and ultimately, ‘where the phage fits in the database’.
What was the purpose for performing a dilution series on your phage?
Thanks for the question Laura!
Serial dilutions were part of the protocol for producing a ‘High Titer Lysate’, or a highly concentrated solution of phage particles. The first step is to scrape phage from our streak purification plate (ensuring we have one new phage with unique and consistent morphology) into a tube of phage buffer. This creates a baseline concentration of phage, which we expect to be really strong and clear all the bacteria when plated onto M. smeg. We perform serial dilutions on that tube, resulting in 5 phage samples with decreasing concentration. When all 5 samples are plated, we expect to see a decreasing amount of plaques on each subsequent plate as the dilution level increases (and the phage concentration decreases). This is ideal because we need a ‘web-patterned’ plate (lots of overlapping plaques with bacterial lawn still visible in between), not a completely cleared plate, to produce our High Titer Lysate and eventually calculate the titer, or concentration of phage particles, in our original phage sample. In the end this is crucial information for archiving the phage and figuring out its infection pattern. Hope this clarifies some things!
Is the M. smegmatis infection indicative of the phage’s ability to infect other bacteria?
Hi Lea! Great question, it definitely could be! M. smegmatis is often used in bacteriophage research as it is very similar to the bacteria M. tuberculosis, for example, which as we know is very pathogenic toward humans unlike M. smeg. This makes it useful to isolate phages which infect M. smeg, because phages can often infect species of bacteria which are very similar to one another genomically and morphotypically. Building a large database of M. smeg infecting phages could benefit research into phage therapies for tuberculosis infections in humans going forward. 🙂