Rationale: Checked NapoleonB’s annotations as a class for mistakes. Started brainstorming ideas for the poster.

Tools:

• DNA Master
• GeneMark
• phage notes

Procedure:

1. Checked for mistakes by scrolling through the frames window in DNA Master.
2. Used GeneMark graph to check that all coding potential was covered.
3. Checked annotations in phage notes.
4. Started brainstorming what belongs on the poster and what format to use.

Results:

NapoleonB’s genes 25-28 covered all coding potential. No corrections were made.

Conclusion:

After brainstorming with the group, it was decided that the methods section should be presented in a flow chart for easy comprehension. Use of columns also would allow for better organization. The color scheme being used should be easy on the eyes. The TEM, gel, and phamerator should be presented on the poster. The poster should also include the final version of the abstract; the names of the researchers,  TAs, and LAs; and a title.

Future Work:

Finish abstract and start designing the poster.

Rationale:

Corrected NapoleonB gene 26 and combined abstracts from other group members for a possible poster abstract.

Tools:

• DNA Master
• NCBI
• PhagesDB
• GeneMark
• HHpred
• phage notes

Procedure:

1. Opened DNA Master and phage notes.
2. Checked for mistakes in NapoleonB gene 26 with GeneMark, “Choose ORF start” window in DNA Master, NCBI, PhagesDB, and HHpred.
3. Corrected the start coordinates.

Results:

The picture below shows the corrected annotation for NapoleonB gene 26.

Conclusion:

The start coordinates were corrected to 24802 bp.  Cooper’s and Lily’s introductory sentences, Nathan’s methods part, and my results sections were combined into an abstract. Working in groups can be a great tool when brainstorming ideas for a presentation; however, groups are not as effective when it comes to writing. One interesting finding placed in the abstract was that NapoleonB is the only AM clustered phage to have a gene with the holin function.  This gene might be the reason why NapoleonB had an extremely high titer relatively fast.

Future Work:

Start individual research projects and design a poster to show at conferences.

In the first four chapters of Anna Kuchment’s The Forgotten Cure, the presented bacteriophage history offers two interesting themes relating to the process of scientific discovery. First, anybody who is passionate enough can participate in research, and second, society and politics will influence the science community’s views of what receives priority in publication.

Cited as the father of bacteriophages, Félix d’Herelle did not have the benefit of a college education. His discovery resulted from his intuition and curiosity. D’Herelle teaches himself and seeks after an unpaid research position. Through his careful observations of locust and stool samples while working as an unpaid assistant, his curiosity drove him to hypothesize why the phenomenon of plaques forming occurred. His desire to answer his hypothesis led him through trial and error in the lab. This resulted in his phage discovery and the development of phage therapy to cure dysentery patients. His curiosity caused him to continue to make effective therapies for Bubonic plague and cholera. Another bacteriophage researcher, Georgi Eliava shared d’Herelle’s same passion. He too observed the plaque-appearing phenomenon and which sparked his curiosity. Unlike d’Herelle, politics had a stronger influence on Eliava’s work. Eliava received government support through funding and expensive science instruments under Lenin. However, Eliava’s work came to an abrupt halt as Stalin’s xenophobia caused the deaths of many Soviet intellects.

One reason why most research goes unacknowledged by the scientific community is that the research is not published. If a scientist does not publish their work, they might as well never have conducted it. Authors such as Sinclair Lewis and Paul de Kruif reeducated the public about the realities of research institutions and practicing physicians. Researchers will rush to publish experiments that were not complete to beat their rivals to receive credit.  D’Herelle’s published work received bad publicity which greatly impacted the spread of phage therapy. As others tried or rush their experiments, they made published papers which argued against his findings. World War II made the scientific community made the military’s needs a priority. Since Fleming’s penicillin seemed more promising and negatively publicity left phage therapy’s effectiveness in question, many physicians turned to antibiotics for treatments.

In conclusion, it surprising what d’Herelle accomplished without a degree. The critical role of publication and the powerful influence of politics on the scientific community is also shocking.

Rationale:

Annotate NapoleonB genes 26-28 to complete the section of genes assigned. Once NapoleonB genome is fully annotated, ideas for what research question should be investigated with this data can be explored.

Tools:

• DNA Master
• NCBI
• PhagesDB
• GeneMark
• HHpred
• Phamerator
• phage notes

Procedure:

1. Conducted a GeneMark on NapoleonB.
2. Determined the stop and start with the highest coding potential for genes 26-28 based off the “Choose ORF Start” window and the GeneMark.
3. Performed a HHpred search on NapoleonB genes 26-28.
4. BLAST was performed on NapoleonB genes 26-28 through both NCBI and PhagesDB.
5. Annotated NapoleonB genes 26-28.

Results:

The following image shows NapoleonB gene 26-28’s annotations.

The following images show the BLAST E-values for gene 26 from the start codon which Glimmer and GenemMark called.

The following images show the BLAST E-values for gene 26 from the new start codon which has the longest reading frame.

Conclusion:

On gene 26, it was determined that the start codon should be moved from what Glimmer and GeneMark called (bp 24817) to an earlier start codon (bp 24802) because the BLAST E-values were smaller, more coding potential would be covered, and this would make the gene have the longest open reading frame. Genes 26-28 had no known function.

Future Work:

NapoleonB’s annotations will be checked and research questions to be investigated will be brainstormed.

Rationale:

Start annotating NapoleonB. Once NapoleonB fully genome is annotated, the process of brainstorming research questions about NapoleonB can begin.

Tools:

• DNA Master
• NCBI
• PhagesDB
• GeneMark
• HHPred
• Phamerator
• phage notes

Procedure:

1. Auto-annotated NapoleonB FASTA file.
2. Conducted a GeneMark on NapoleonB.
3. Determined the stop and start with the highest coding potential for gene 25 based off the “Choose ORF Start” window and the GeneMark.
4. Performed a HHPred search on NapoleonB gene 25.
5. BLAST was performed on NapoleonB gene 25 through both NCBI and PhagesDB.
6. Annotated NapoleonB gene 25.

Results:

The following image shows NapoleonB gene 25’s annotation.

The following images were examined to determine whether or not there was enough supporting evidence for a function.

HHPred Results

NCBI BLAST Results

CDD Results

BLAST PhagesDB Results

Phamerator Comparison

Conclusion:

A difficult call occurred when determining whether or not there was enough supporting evidence for gene 25 to have a function. HHPred called a holin function with a 99.92 probability. CDD also called the holin function. Both BLAST results showed a low percent alignment, but the E-values were acceptable. On Phamerator, none of the other AM cluster genes have the holin function labeled on them. Gene 25 was annotated with the holin function.

Future Work:

Annotate NapoleonB genes 26-28.

Rationale:

Become familiar with phage notes, a Google spread sheet form designed to lower the number of mistakes when filling out the SEA-PHAGES template, and to peer check each other’s genes to further lessen the number of mistakes.

Tools:

• DNA Master
• NCBI
• PhagesDB
• GeneMark
• HHPred
• phage notes

Procedure:

1. Copied and pasted Elesar genes 54 and 56 into phage notes.
2. Performed an HHPred search on Elesar genes 2 and 3.
3. BLAST was performed on Elesar genes 2 and 3 through both NCBI and PhagesDB.
4. Conducted a GeneMark on Elesar.
5. Annotated Elesar genes 2 and 3.
6. Compared Elesar genes 2 and 3 annotations with peer’s.

Results:

The following image shows Elesar genes 54 and 56 annotations filled into phage notes.

The following image shows the checked Elesar genes 2 and 3 annotations in phage notes.

The annotations were completed except for the starterator section.

Conclusion:

Both phage notes and peer checking lowers the number of mistakes in the template.

Future Work:

Start annotating NapoleonB.

Rationale:

Added on function (F) and supporting information for function (SIF) for Elesar genes 54 and 56.

Tools:

• DNA Master
• NCBI
• PhagesDB
• HHPred

Procedure:

1. Performed a HHPred search on Elesar genes 54 and 56.
2. Performed a BLAST through NCBI and PhagesDB on Elesar genes 54 and 56.
3. F and SIF portions of Elesar genes 54 and 56 were filled out.
4. These annotations were saved.

Results:

The following image shows Elesar gene 54 HHPred results.

The following image shows Elesar gene 56 HHPred results.

The following image show Elesar gene 54 fully annotated.

The following image show Elesar gene 56 fully annotated.

Conclusion:

Both the functions of Elesar gene 54 and 56 remain unknown.

Future Work:

Start annotating NapoleonB.

Rationale: Finish going over genome annotation temple and practice annotating with Elesar gene 54 and 56.

Tools:

• DNA Master
• NCBI
• PhagesDB
• GeneMark

Procedure:

1. The BLAST-Start, Gap, LO, and RBS portions of Elesar gene 1 were filled out.
2. BLAST was performed on Elesar gene 54 through both NCBI and PhagesDB.
3. Annotated Elesar gene 54.
4. BLAST was performed on Elesar gene 56 through both NCBI and PhagesDB.
5. Annotated Elesar gene 56.
6. These annotations were saved.

Results:

The following image shows Elesar gene 1 fully annotated.

The following images show the GeneMark for Elesar between 36000 to 40000 bp.

The following images show the BLAST results for Elesar gene 54.

The following image shows Elesar gene 54 fully annotated.

The following images show the BLAST results for Elesar gene 56.

The following image shows Elesar gene 56 fully annotated.

Conclusion:

For both Elesar genes 54 and 56, they were most similar to another gene from Elesar. PhagesDB seems to have more BLAST results with smaller E values. Although PhagesDB is a smaller database, it seems to be more helpful than the NCBI database since it has more phage DNA.

Future Work:

Continue to practice annotating with Elesar and learn about phamerator.

Rationale: Go over guiding principles along with other analysis tools, such as GeneMark and BLAST and start to fill in the notes template.

Tools:

• DNA Master
• NCBI
• PhagesDB
• GeneMark

Procedure:

1. In DNA Master, the main window, the frames window, and the choose ORF start window were opened.
2. BLAST was performed on Elesar gene 1 using both NCBI and PhagesDB.
3. The SSC, CP, and SCS portions of the template for Elesar gene 1 were filled out.
4. This annotation was saved.

Results:

After performing BLAST through both NCBI and PhagesDB, it was determined from the high E value that there was no significant alignment found. The images below show these results.

It was determined through GeneMark that the start choice (84 bp) predicted by Glimmer and GeneMark was not the LORF (longest open reading frame). At 45 bp, all possible coding potential was covered as shown in the GeneMark graph below.

The following screenshot shows what parts of the template where filled in along with the values in the choose ORF start window.

Conclusion:

Although it is rare to see TTG as a start codon, it can be a start codon. The start choice predicts from Glimmer and GeneMark are not always accurate.

Future Work:

The result of Elesar gene 1 template will be filled out.

Rationale:

Insert template and learn more about basics of annotating.

Procedure:

1. Opened DNA Master.
2. Changed preference to insert template during auto-annotation.
3. Auto-annotated Elesar.

Results:

The template was successfully inserted and auto-annotation was successfully performed with the template on Elesar as shown below.

Conclusion:

No difficultly was faced when auto-annotating Elesar. The conceptions learned today in lab were reading frames, how to calculate gaps and overlaps, and when a gap is required. A gap of at least 50 base pairs is required when the promoters are going in opposite directions.

Future plans:

Start analyzing the auto-annotation of Elesar and performing BLAST on Elesar.