April 12

Transposon Searching 4/8/2019

Title: Transposon Searching

Date: 8 April 2019

Rationale: We will continue to search for a purpose or additional information that can define the 45-bp sequence (with 6-bp palindromic flanks)

Procedure: Upon BLASTing the sequence again on PhagesDB, it was found that 11 out of 14 phages contain the sequence, with 2 phages, NapoleonB and Kardesai, containing 2 copies of the sequence. The phamerator maps were inspected and more research was done to find literature that could support the finding being a promoter or transposon, or otherwise.

Results/Observations: All phages, except for the 2 mentioned above, that contained the sequence had a gene sequence consensus of a gene in pham #37812 followed by pham #1350. However, the 2 phages that contained a copy of the sequence had a gene in pham #37812 followed by a gene in pham #620. It is worth noting that only NapoleonB and Kardesai have copies of this gene phamily.

Conclusions/Next Steps: All of the sequences will be inspected to look at the flanking regions as well as searches conducted on the internet to find programs that may be able to predict transposons or promoters.

 

Category: Cooper Johnson | LEAVE A COMMENT
April 12

Check in and Symposium Presentation

4/10/19

Rational:

To evaluate the project’s progress as well as my own contributions to the project. Also to create an outline of the presentation for the Symposium on the process behind our research. Comparisons between the different protein stuctures were also done

Procedure:

  • Each individual filled out the form for the QTM evaluation
  • After the print outs of the different phage’s endolyins and holins were compared to see similarities and differences
  • The uses of Jmol were also explored in more detail to be able to use it more efficiently
  • The group created a shared presentation and created an outlne for the presenation

Fig.13 – This image shows the original title slide for the presentation

Fig.14 – This image shows the amino acids that are part of the catalyic site of Elesar’s endolysin

Conclusion:

We were able to better understand Jmol so that we can better use it as a tool. We were able to see how similarly or dfferently the structures of other phage’s holin and endolysins were. We were also able to create a outlne for the presentation. Next we will find where the catalytic site is on other phages and finish the presentation for the Symposium on Friday.

Category: Emily Balint | LEAVE A COMMENT
April 12

Hard Data (4/8/19)

Rationale: The rest of the hard data was entered into the Google Excel sheet.

 

Tools:

  • Personal Computational Device
  • Google Excel
  • Phages DB

 

Results:

  • Data from PhagesDB was entered into the Google Excel sheet in groups separated into GC content, gene length, etc.

Conclusion:

The rest of the data was entered into the sheet.

 

Future Plans:

To begin looking at protein structures in the AM cluster

Category: Sabin Patel | LEAVE A COMMENT
April 12

Source Submissions

4/8/19

Rational:

To find five sources that give background information to the research project. Also, to find the catalytic site for the endolysin.

Procedure:

  • Used the folder of sources collected
  • Chose five sources that were background information
  • Found a tool that could show where certain amino acids are found
  • Used Jmol to find the amino acids related to the catlytic site
  • Found the possible catalytic site

Fig.12 – This image shows the section (HLH) that is part of the catalytic domain in endolysins

Conclusion:

The five bacground sources that were needed were found. We were also able to find a tool that showed where specific amino acids were found whch was not something that could be done on RaptorX. Next lab we will find the catalytic sites of other phages and figure out how to better use Jmol

Category: Emily Balint | LEAVE A COMMENT
April 12

4.10.19 IRP

Rationale:

To continue working on our IRP, by further looking at background research, protein structure, and multiple sequence alignments.

Procedure:

  1. The structure of gene 93 was looked at on TMHMM, in roder to provide evidence that it is a transmembrane transporter protein.
  2. MSA’s were run in order to show phylogenetic relationships.
  3. Further research was done into the pham changes.

Results/Data:

According to TMHMM gene 93 is indeed a transmembrane protein. The pham we were looking at on Monday was split, eliminating a lot of the confusion that was present on Monday. The new MSAs conducted show much more similarity between the different phages/bacteria.

Conclusions/Next Steps:

We can conclude that gene 93 is an NMT. We can also conclude that there was an apparent error in the pham system, as it is now fixed and makes a lot more sense. We can also start creating a phylogenetic tree based on our MSA data. The next step will be to continue investigating NMTs and NMN, along with conducting more MSA’s on more phages and bacteria in order to increase our understanding of the phylogenetic relationships and origins of the NMT gene in NapoleonB.

Category: Nathan Newton | LEAVE A COMMENT
April 12

4.8.19 IRP

Rationale:

To continue working on our IRP by looking into the NMN pathway and NMT related proteins in other organisms and phages, we will also run MSA’s in order to determine phylogenetic relationships.

Procedure:

  1. I conducted further background research into NMT and NMN.
  2. We looked at other phages in the gene 93 pham.
  3. We investigated the differences in NMT, NRT, and NRTransferase, using both literature and HHPred.
  4. We ran multiple sequence alignments.

Data/Results:

We found that there may be apparent errors in either the pham organization or gene calling of other phages in regards to NMT, NRT, NRTransferase. We also found some relations between the various phages and bacteria compared via MSA but some error may have been involved, but it does appear that there are relations.

Conclusions/Next Steps:

We can conclude that there are relations between gene 93 in NapoleonB and similar genes in other organisms/phages, which will ultimately allow us to find phylogenetic relationships and help us to determine the origin of gene 93. We can also conclude that something weird/incorrect is happening with the pham organization and the gene calling of other phages. Our next step will be to continue investigating phylogenetic relationships and running MSAs, while also looking at protein structures in order to provide evidence/support.

Category: Nathan Newton | LEAVE A COMMENT
April 12

04/10/19 JMOL and Catalytic Region Hypothesis

Rationale:

The purpose of today’s lab was to further our knowledge and ability of JMOL to better understand our protein structures.

Tools:

  • JMOL
  • JMOL Instruction Guide

Procedure:

  • Began by familiarizing ourselves with the commands used in JMOL to produce effective results.
  • Once completed, all Histidine, Tyrosines, and Aspartic acids were labeled in the protein with the colors red, blue, and yellow respectively.
  • Following this, wireframe was turned on to allow us to see in which direction these residues faced, allowing us to visualize which residues interacted with each other best.
  • Next, the group hypothesized which residues would best fit our catalytic region based off motility, orientation, and distance from the 4 histidines that are essentially bound in place.

Results:

These are the proposed catalytic regions found in 3 different phages from 3 different clusters (AM, FE, and FF)

NapoleonB Endolysin

Corgi Endolysin

Elesar Endolysin

Conclusions:

It can be concluded that these catalytic domains are conserved well throughout the M23 Peptidase family and Arthrobacter phages. The aspartic acid as well seems to be structurally conserved as every endolysin examined contained an acidic residue of aspartic acid on the neighboring beta sheet, which suggest it plays a role in the reaction.

Next Steps:

The next steps for this project are to use the knowledge of these endolysin proteins to help call function to NKF genes in different Arthrobacter phages.

Category: Gabriel Andino | LEAVE A COMMENT
April 12

04/08/19 JMOL and Catalytic Domain

Rationale:

The purpose of today’s lab was to analyze the RaptorX results generated for the phage holin and endolysin proteins and compare the different structures.

Tools:

  • RaptorX
  • PubChem
  • Jmol

Procedure:

  • Initially began with looking at the RaptorX PDB images generated, however they did not give much information in regards to the catalytic regions especially as we had no information regarding specific residues.
  • Tried switching to PubChem, and though we were able to spin the protein, there was still no information regarding the actual amino acids.
  • Eventually, downloaded JMOL, which offered the information we were looking for, and we were able to find the Histidine, Leucine, Histidine region of the catalytic Domain.

Results:

Histidine, Leucine, Histidine Catalytic Region

Elesar Endolysin

NapoleonB Endolysin

Conclusions:

The supposed catalytic regions were found, however there is much about this program that is unknown and there is definitely a learning curve to it. The tyrosine and acidic residue locations are still unknown in the protein.

Next Steps:

Figuring out how to utilize JMOL effectively is number one priority as it has the ability to give incredible amounts of information regarding the protein structures above. The missing tyrosine and acidic residues still need to be identified in the protein.

 

 

Category: Gabriel Andino | LEAVE A COMMENT
April 12

4/10 ~ Continued research for Independent Research Projects

Rationale: Continue to gather data to answer the scientific question regarding preferred start codons and the relationships that can be drawn between clusters and phages within clusters

 

Materials:

  • DNA Master
  • Google SpreadSheet
  • PhagesDB

 

Procedure:

  • Loaded up the FASTA files from PhagesDB of the AR phage and annotated them in DNA Master
  • Recorded the start codons of each gene and calculated the percentage of each start codon (ATG, GTG, TTG)

 

Observations:

  • The preferred start codons of phage KBurrousTX

    The preferred start codons of phage Linus

     

Conclusion/Next Steps: Similar to the AM cluster, there were patterns within the AR cluster which is unique to the phages found within the cluster. In the examples of phage KBurrousTX and Linus, the last five genes of the phages preferred an ATG-GTG-ATG-ATG-ATG pattern which was found throughout all the phages in the AR cluster. The next steps would be to calculate the preferred percentage of the start codons and observe if there is a correlation between clusters or phages in the clusters.

Category: Justin Yu | LEAVE A COMMENT
April 12

4/8 ~ Research for Independent Projects

Rationale: Find date for independent research projects which support the scientific question

 

Materials:

  • DNA Master
  • PhagesDB
  • Google SpreadSheet

 

Procedure:

  • Opened DNA Master and opened FASTA files of phages in AM cluster
  • Recorded each start codon for each gene
  • Determined the preference percentage of each start codon (ATG, TTG, GTG)

 

Observations:

NapoleonB with the preferred percentages of each start codon

Mudcat with the preferred start codon of each gene

 

Results/Next Steps:

  • When observing the preferred start codons, there was a pattern in the last four genes of the phages in the AM cluster. They all preferred a GTG-GTG-ATG-ATG for their last 4 genes, which may show that they code for similar genes. This discovery can help other scientists help characterize and cluster their phage earlier in research. Will continue to find the preferred start codons of the AM cluster as well as the AR cluster
Category: Justin Yu | LEAVE A COMMENT