The purpose of this lab was to make our poster and figures as well as to refine our presentation.

Methods

1. Create a shared google powerpoint slide with a modern background.
2. Make sections for each part of a research paper abstract methods etc.
3. Write a short summary of what should go in each section including important points and figures.
4. Using the screenshots you have collected of your work create the figures you would like to use with labels and captions.
5. Find sources for background information on your topic.
6. Begin writing the abstract and conclusion.
7. Check that the fonts are large enough and have enough contrast to be clearly readable.
8. Check that fonts and outlines are uniforms.
9. Organize the overall sections of you slide so that there is a logical progression of your project and so that the results are focal and easy to understand.
10. Make sure your slide is inviting/intriguing and not too wordy.
11. Complete each section and have each member of the group review the slide and make final adjustments.
12. Decide who of the group will present what and address any common questions that may be asked.
13. practice presenting with your group.
14. download the slide as a PDF and submit it to your instructor.

Results:

We were unable to complete all the steps and we needed to work on our abstract, conclusion, and works cited.

Conclusion:

Making and taking a quality poster takes more than one class period.

Next Steps:

The group scheduled a meeting time outside of class to finish the poster.

Kiersten Scott

4/15/2017

The purpose of this lab was to locate holin proteins in phages similar to Timinator, to record these phages and proteins, and to submit the genes products of these proteins to the Swiss Model and to RaptorX for further analysis.

Kiersten

1. Found fully annotated genomes in the same Holin pham as Timinator and created a gene map.
2. Identified possible Holin proteins in other phages, compiled a list of said phages as well as the locations of their Holin proteins.
3. Recorded a picture of the phamerator output to use for the final poster in order to show the relationship of the phages.

Teresa

1. Download, saved, and opened phage genomes in DNA Master.
2. Submitted holin gene products to Swiss Model and Raptor X.
3. Recorded Swiss Model output reports.

Kirsty

1. Helped with both Kiersten and Teresa’s tasks.
2. Curated the group data that was already collected and made a list of data that we still needed.
3. Began formatting work on the final poster.

Results

We all worked together as a team to finish collecting data for out project.

Conclusion

There are some visible similarities in Holin gene products for Arthrobacter phages. Although we are waiting on more definitive information from RaptorX to accept or reject our hypothesis.

Next Steps

During the next lab we with use the information we have gathered so far to make a poster.

Kiersten Scott

04/02/2017

The purpose of this lab was to being to execute our research project into holin proteins.

Procedure:

1. Using the functional annotation techniques we learned in labs 1-9 Kirsty and me checked the functions of the suspected endolysin and holin genes for the phages Timinator and Lore.
2. We found the gene products of each suspected gene, and after we resolved any questions about the annotation we ran the gene products through Phages.db, NCBI, and HHPred
3. Meanwhile, Teresa created a phamerator output to compare phages with the same pham numbers as the holin genes in Timinator and Lore.
4. After we confirmed the function of our genes I ran two suspected genes through the Swiss Model program.

Results

We made good headway our project and found encouraging information that we recorded to report later on.

Conclusion: I hope to learn more about the Swiss Model program so that I can better understand it’s output. During the next lab, I hope to uses Teresa’s data to find more genes in annotated phages to run through the model to hopefully compare in the final stages of our project.

Next Steps

We need to continue working with phamerator and investigate the structure analysis tool RaptorX

Kiersten Scott

03/27/2017

The purpose of this lab was to decide our line of research for our research as well as to make a plan and delegate roles to complete the project.

Method

1. We analyzed our possible research questions from last week.
2. We looked up primary sources about our questions.
3. We talked with each other and decided to investigate the holin protein.We  found this protein intriguing because it is central in causing the lysis of a bacteria.
4. We looked through the annotations of Lore and Timinator and found that both had an identified holin protein. Particularly genes 15, 18, 17 in Lore and genes 20, 27, and 28 in Timinator.
5. We ran both genomes through Phamerator and recorded their pham numbers.
6. We looked through ExPASy and found possible tools, like Abalone and the Swiss model, we could use to visualize the holin protein.

Results

We have created a plan of action for our project and we intend to research differences in holin structure across Arthrobacter phages, particularly the phams of Timinator and Lore.

Conclusion

The holin protein is interesting because it is highly specific and variable among phages. We hypothesize that we will find similarities between the holin proteins of Arthrobacter phages. This is highly important in understanding how different phages lyse their host bacteria and has many applications.

Next Steps

We need to reannotate the possible holin genes and find similar Arthrobacter phages to compare it to

Possible Sources

https://www.ncbi.nlm.nih.gov/pubmed/11018145

https://www.ncbi.nlm.nih.gov/pubmed/10707065

https://www.ncbi.nlm.nih.gov/pubmed/7669346

https://books.google.com/books?id=nozn1K8tcRsC&printsec=frontcover&dq=the+bacteriophages&ei=WzuIS5_IMY3ulQTF5Ly2DQ&cd=1&hl=en#v=onepage&q=10%20phage%20lysis&f=false

Kiersten Scott

3/18/17

Th purpose of this lab was to complete the annotation of a second genome (timinator) in order to refresh annotation skills and to stoke the formulation of phage hypotheses to be tested in later labs.

Procedure

1. Complete Timinator annotations according to the guiding principles and using the steps and experience provided by labs 1-7.
2.  Note any unusual annotations and made individual notes for Timinator’s cover sheet.
3.  Then form a three person experimental group.
4. Search through Phagesdb information on phage proteins, novel phage research programs, and previous lab notes.
5. Note any intriguing developments or questions you have.
6. After each member of the group has completed this step talk to one another about the avenues of research that you have identified.

Results

Phage annotation has become much easier and I can’t wait to decide on a hypothesis and to apply my skills.

Conclusion

My group arrived at several interesting questions and we will decide in our next lab which one we will examine as well as how we intend to do so.

Next Steps

Our group needs to decide on a question to research and begin to refine our hypothesis

Timinator notes-200cpfs

Kiersten Scott

February 23,2017

The purpose of this lab was to get a broad overview or the Lore annotation and to discuss any remaining points of concern before beginning the submission process.

Procedures

For this lab, each group had a presentation where they showed their annotated genes, described their functions and defended any changes that they made. My group annotated genes 14 and 15 without changing the original calls, or finding any known functions. While gene 14 was straight forward, gene 15 had to have its start site defended. This was because there were a few alternative start sites upstream, although these starts would have allowed a longer reading frame they would have created a significant overlap and disagreed with several other lines of evidence.

Submission

After each gene questionable gene is reviewed the submission process will begin this includes:

1. validating the genome
2. renumbering the genes
3. recreating the documentation
4. reblasting the genome
5. double checking the blast matches
6. making a duplicate
7. make an author list
8. write a cover list

Conclusion

The overall annotation of Lore is solid however there are still some points of concern regarding genes 3,9, and 21. Other than these points the q:1 s:1 NCBI BLAST data must be confirmed before the submission process begins. Overall, the past few weeks have given us the tools we need to annotate and the class is nearly ready to move on to another genome.

02/19/17

Kiersten Scott

The purpose of this lab was to identify and address any areas of concern on the annotations of the Lore genes 14 and 15.

Method

The method of this lab builds on the method from the last three labs.  By using the Glimmer, GeneMark, PhageDB, NCBI, HHPred, Phamerator, and Starterator programs I was able to lead a  fellow student through the process of annotating a gene.

Methods

First, we opened the lore genome in Glimmer GeneMark, and Phamerator to assess coding potential and whether the three programs agreed with the chosen start site. Then using the Pham numbers we ran the genes to Starterator. After we felt that we had collected enough evidence we selected a start site and checked its Shine Delgarno reading to see if our start site was acceptable or if a better option existed. Once we were satisfied with the results we recorded the start site, the gap or overlap, and whether the gene had the longest open reading frame. The next step was to identify any possible function so we ran the protein products of the genes through NCBI, PhagesDB, and HHPred and recorded the best possible hits.

Results

Both of us were able to successfully annotate a gene as well as address a region of uncertainty with the start site of gene 15. After reviewing our evidence we decided to use a gene with a shorter open reading frame with aligned best with the calls from Starterator our BLASTs and the Shine Delgarno score.

Kiersten Scott

02/08/2017

The purpose of this lab use to learn how to use and read the program Starerator, and to incorporate it’s output into a full genes annotation

Methods:

1. Open virtual box and log in.
2. Open Phamerator and wait for it to update if necessary.
3. Using the phage search tool find the phage of interest and select Map.
4. Find the gene(s) of interest and record their Pham number(s).
5. Close the output and Select Starterator from the Virtual Box.
6. Input the Pham number and record the recommended start site for the gene.
7. Compare the recommended start site with start sites proposed by Glimmer and GeneMark and decide whether or not to change your start site
8. Record whether or not you agreed with Starterator’s call in your gene annotation.
9. Complete the rest of the annotation looking back to past methods if necessary.

Results

During this lab, I fully annotated two genes in the Lore phage from 9801 bp -11,200bp. So far I haven’t found a function for any of my annotated genes but I am excited to keep looking as well as to master gene annotation. The results of my annotating can be seen below

.

One of my Starterator outputs.

My full annotations

Kiersten Scott

Feb, 1, 2017

The purpose of this labs was to understand a new tool, Phamerator, and to incorporate this tool into a working gene annotation.

Methods-Phamerator

1. Click on Virtual box>Sea2017>phamerator. Search for the genome of interest and also choose 4-6 closely related genomes before clicking Map.
2. This will give you an output that can match genes over several genomes. Record the Pham number, members number, and cluster.
3. Check to see if a function has been identified in your gene of interest or the genes around it. If so record.

Methods-annotation continued

To make a proper annotation you must fill each of these fields: SSC: CP: SD: SCS: Gap: Blast: LO: ST: F: FS

Follow the instructions from last week regarding the blanks SSC: CP: SD: SCS: Gap: and LO, this leaves Blast: F: and FS:

1. Blast: Go to the NCBI and Phagesdb and enter you suspected gene in BLAST. Record the top hits, their e-values, and the suspected function if there is one.
2. F: Using the Blasts from NCBI, Phagesdb, HHpred, and Phamerator consider the tops hits you received and the synteny of your gene and make your best guess to the function of the protein if there is one.
3. FS: List the resources that you used to make your functional prediction NCBI, Phagesdb, HHpred, and Phamerator.

Conclusion:

My group worked on annotating gene 22 in the Link phage whose details are included in the information below.

NCBI output                                                       HHPred output

Phagesdb Output                                              DNA master and annotation

This lab built heavily from the material and tools used and detail in previous lab reports. By keeping a detailed report of each step the annotation process can be repeated until it is a fairly simple process.

Kiersten Scott

01/27/17

The purpose of this lab was to establish methods to accurately annotate a genome.

Method

To make a proper annotation you must fill each of these fields: SSC: CP: SD: SCS: Gap: Blast: LO: ST: F: FS: ST

1. Upload your genome into DNA Master and GeneMark.
2. SSC (start stop coordinates) Look at the gene holistically and write the start and stop coordinates and their orientation (forward/backward) in the annotation. Note that these numbers may change as the annotation is completed
3. CP (coding potential)-Check that your start coordinates cover all of GeneMarks’s coding potential if not you may need to adjust the start of the gene. Once you have chosen the farthest possible start write that the coding potential is covered in your annotation.
4. SD (Shine Delgarno)- Using a shine delgarno window check that your open reading frame has a low score. If your gene has a high score you may need to adjust the start coordinates of the gene. Once you are satisfied with the score write it in your annotation.
5. SCS (start choice source)-Check that the Glimmer calls agree with GeneMarks’s coding potential. Once you are satisfied with the two reports alignment write that the two reports agree in your annotation.
6. Gap (state of ends)-Check the gap or overlap of the gene making sure that overlap is no more 30 basepairs, 50 between two opposite strands. Take note if the Gap is to wide or small there make be and extra gene of the may be part of an operon. Calculate the gap or overlap and add it to your annotation.
7. BLAST—(more information later).
8. LO (longest ORF (open reading frame)) Check once more that the gene is as long as it can be while fitting in with the general scheme of the total genome (it shouldn’t be the only inverted gene in the genome etc.)
9. F —, FS—, ST—(more information later).

Future Thoughts

By following and practicing these steps students can become more confident in their ability to annotate genomes as well as use what they have learned so far to make inferences about the genes they annotate.