Rationale: Now that data has been gathered for the independent research, I need to find a good way to visually and numerically display this data. On top of that my group worked on making an abstract  to submit to CURES in bio.

Tools: MEGA, Google Drive, Jmol

Procedure:

1. Using  Jmol and MEGA to visually demonstrate the protein structures of different phages in the supercluster and show the differences from each and created a phylogenetic tree to display that data.

2. Worked on the rough draft of the abstract from before and made sure that the tense was correct, as it was active voice before.

Results: Data showed that the AM cluster is very closely related to the EL cluster and the phylogenetic tree showed the closeness in the DNA sequence of the members of the supercluster as to other arthrobacter phages.

Conclusions and Future Work: I now need to complete putting my data in a presentable format and prepare for CURES in bio.

3. As a scientist, describe the main experiment you would like to see performed before phage therapy is approved for human use. What are the risks involved with using phage therapy?

I would like to see tests done on the human response to therapy. Many phage therapies could evoke an allergic reaction as the immune system mounts an attack on these foreign bodies. Furthermore, how would the kidneys and liver function differ, especially in patients with conditions weakening these filtration systems, when trying to filter out the remains of the bacteria cells and phage particles. This includes the effects of endotoxins, in which people could enter septic shock causing much fatality. As with most therapies that could involve a toll on either the kidneys or the liver, enzymes would have  to be monitored closely during the entire time of clinical testing. In addition dosage of phage therapy would have to be taken in to account before it reaches the point that the immune system will start mounting  a defense against the foreign particles.

Rationale: I’m continuing the process described in the previous post to try to discover new superclusters.

Tools: PhagesDB, Phamerator

Procedure:

1. Looked through DNA primase phams and looked through phams within the AK cluster.

2. Noticed a plateau in the pham cluster members similar to when looking at the pham cluster members of NapoleonB.

3. Recorded my findings.

Results: The clusters that contain  a lot of similar synteny and similar phams:

• AK
• EA7
• EJ

Conclusions and Future Work: Now that I’ve found a couple instances of what could be described as superclusters I need to find a numerical way or visual way to display my findings of these common syntenys and genes.

Rationale: After talking to Dr. Pope at the phage emporium I decided the best move is to look at modules containing genes that originate in bacteria to find more superclusters. By finding more superclusters I can compare against the one I’ve already found to make the claim that these are a thing.

Tools: PhagesDB, Phamerator

Procedure:

1. I started by searching through phams in phagesDB that are called as cas4 exonucleases.

2. I looked at the phamerator maps of multiple clusters containing a cas4 exonuclease and tried to find patterns in the gene synteny.

3. Discovered another instance of what might be a supercluster that contained a module of DNA primase followed by a DNA polymerase.

Results:

Clusters found to contain similar synteny and the 45806 primase module:

• AZ
• BB
• BB1
• BB2
• BJ
• BL
• EB
• EH

Conclusions and Future Work: This method of discovering new superclusters seems to work quite well I’m going to continue looking for more superclusters so I can compare them against each other.

Rationale: My partners are working on putting together the phylogenetic tree and looking more in to the  differences between the  members  of each cluster in the supergroup. So, I am going to look more in to  the  different transmembrane  proteins to  try to find a pattern.

Tools: Jmol,  RaptorX

Procedure:

1. After running a member of  each cluster’s transmembrane  protein through raptorX I viewed each protein in Jmol.

2. Noticed a pattern of alpha helix cluster and beta pleated sheet section.

3. Noted my findings  and  tried to find correlation with clusters outside of the group of clusters we are currently looking at.

Results:

Conclusions and Future Work: I will be working more on trying to correlate the  findings with out  groups  to provide a comparison against which to make my claim that there is some kind of relationship among all these clusters.

Rationale: Since I was not able to find a relationship between gene 61 and 62 using protein-protein interaction I decided to go back to looking through NCBI and CDD to find evidence for a function for this gene.

Tools: BLASTp, Jmol, RaptorX, CDD

Procedure:

1. Blasted the gene 61 of NapoleonB against actinobacteria.

2. Resulted in  a hit against streptomyces which I looked more into the gene it hit to.

3. Located 3 conserved domains within the streptomyces  gene.

4. Lined up the conserved domain location to  the location that the NapoleonB  gene  matched with the streptomyces gene.

5. Found that the conserved domain G5 lined up  with the gene from NapoleonB.

6. Analyzed the structure of a G5 domain and compared to the predicted structure of  gene  61 and  found that  they are extremely similar.

Results:

Conclusions  and Future  Work: This  most likely  means  that this gene was  just  picked up at the same time as the  DNA polymerase. Now we are going to  work on  putting together a phylogenetic tree mapping the members of this group of  clusters. Also I  will start looking for other instances of groupings of clusters.

Rationale: Since Arcadia’s genome is already full annotated I’m using it as an  example genome for studying this group of genes. I’m going  to be figuring out if there are any interactions between these proteins to give insight on their function.

Tools: RaptorX Structure Predict, RaptorX DeepAlign, Jmol, TMHMM

Procedure:

1. Ran the protein  sequence of  Arcadia’s genes 59-61 through the  structure prediction tool of RaptorX to discover what the protein products of the genes would look  like structurally.
2. Ran different combinations of each protein product with each other to see if there is a possibility of the genes interacting with each other using DeepAlign.
3. Visually looked  at each protein product using Jmol and found that gene 59 looks like a transmembrane protein.
4. Ran the protein sequence through TMHMM and found that it is in fact a transmembrance protein.
5. Ran TMHMM for other proteins of members of the supergroup with pham 19129.

Results: DeepAlign results were >.3 therefore there is a greater than 90% probability that there are no interactions between these three proteins.

TMHMM for KeaneyLin

Conclusions and Next Steps: Most likely the proteins don’t interact with each other. However, most of the proteins all seem to be transmembrane proteins so their might be more to look in to there. This protein also seems to be a determining factor of which cluster the phage is in. I will continue looking in to interactions by visually comparing the DNA polymerase and the gene before it with proteins that normally interact with DNA polymerase or replication in general.

Rationale: We spent the time today in lab practicing our presentation for URSA, so that we could seem competent in our understanding of our  research.

Tools: N/A

Procedure:

1. Took turns practicing presenting.

2. Explained to each other what are ways we can improve our presenting.

Results: N/A

Conclusions and Next Steps: Next we will begin working on our independent research.

Rationale: We are beginning to look in to the question and building a plan for moving forward with looking into this research question.

Tools: NCBI Blastp, Word, Phamerator

Procedure:

1. Explored more into the different clusters that contain the pham 19129, and found not all of the clusters infect arthrobacter.

2. Refined and reworded the research question.

3. Blasted the amino acid sequence for the DNA polymerase in NapoleonB’s genome against actinobacteria. Found it hit to Streptomyces.

4. Made a plan for conducting the research going forward.

Results:

Blast results for the DNA polymerase in NapoleonB’s genome against actinobacteria.

Conclusions and Future Work: Now that we can see some sort of start point with the streptomyces the plan is to analyze the differences between the hosts and blast the genes we found similarities in against the different hosts. From there we can compare the slight differences in amino acid sequence and nucleotide sequence in the cassette that contains the three shared genes.

Rationale: In order to move forward with independent research we must first think of a question we want to test. Therefore, we brainstormed some questions that seem interesting for us to test.

Tools: Phamerator, Word

Procedure:

1. Explored the phamerator map looking for interesting things about NapoleonB.
2. Found an interesting pattern between NapoleonB’s DNA polymerase gene and other phages that have the same pham DNA polymerase.
3. Found patterns in the synteny between members of the 19129 pham.
4. Developed question based off the found pattern.

Results: N/A

Conclusion and Future Work: There seems to be a gene right before the DNA polymerase that always shows up in these clusters in the same spot. We must discuss as a group whether this is a good idea to pursue as  an independent research question.