Rationale:

Genes from NapoleonB were assigned and the rest of the lab was given to annotate the four assigned genes.

Tools:

• Personal Computational Device
• DNA Master
• NCBI
• PhagesDB
• GeneMark

Results:

• After the genes were assigned, gene 33 was viewed on DNA Master to determine if the opening reading frame had the most coding potential if the second biggest opening reading frame had the most coding potential.
• To determine this, the base pairs for the second opening reading frame was changed and viewed on GeneMark and compared with the coding potential of the original open reading frame. It was concluded that the coding potential for the original open reading frame was best because it covered all the coding potential

• The opening reading frame was reverted back to its original frame and the product was used to compare the sequence with other sequences in databases such as NCBI, HHPred, and PhagesDB.
• With these databases, other parts of the annotations were determined in order to form the complete annotation.
• The annotation for gene 33 was

Conclusion:

Upon the assignment of the genes of 33-36, the annotation of gene 33 began. Because there was an overlap between gene 32 and 33, the open reading frame was shifted to the second best; however, it was determined that the original reading frame was the best because it covered all the coding potential, unlike the second reading frame. After the best reading frame was determined, the different parts of the annotation were found from databases such as NCBI, GeneMark, HHPred, PhagesDB, and DNA Master. The function of gene 33 was found to have no known function by NCBI, HHPred, and PhagesDB. The annotation for gene 33 was completed.

Future:

In the future (2/13/19), genes 34-36 are to be annotated and annotation of gene 33 is to be checked.

Rationale:

The purpose of today’s lab was to understand how to annotate supporting information of function (SIF) using a variety of databases such as NCBI and HHPred. The information that was given off each database was explained and taught how to use the information to create an annotation. Towards the end, the previous annotation was expanded upon for practice with the addition of SIF-BLAST (NCBI and HHPred.)

Tools:

• Personal Computational Device
• DNA Master
• NCBI
• HHPred

Results:

• HHPred was introduced and was used to demonstrate how to use the information for annotations for evidence.
• NCBI was then used to annotate for SIF-BLAST (NCBI)
• After the annotation, a practice was done on the previous annotations that were assigned (Gene 46 and 47)

Conclusions:

After the introduction of HHPred which provides evidence of a match between two different sequences, NCBI was then used for the same purpose. After the lesson, some practice was done with the assigned genes 46 and 47.

Future Plans:

Other databases will be shown to provide further evidence of the match between the sequences.

Rationale:

The purpose of today’s lab was to get familiar with other databases which provide further evidence of the function of the assigned gene. Along with this, databases which displayed the location of tRNA was introduced as another piece of evidence. The complete annotation of gene 46 and 47 for phage Elesar was completed.

Tools:

• Personal Computational Device
• DNA Master
• HHPred
• NCBI
• PhagesDB

Results:

• After the introduction of the databases for tRNA, PhageNotes was introduced as a program to create a complete annotation of all the data was put in.
• Instructions were given to put in all the information for the assigned genes and to fill in any information was that was not placed.
• When the annotation was checked, several changes were made to the gene in the span of two days which resulted in changes made to the RBS of the previous annotation.

Conclusions:

After the lesson of the databases that were used for tRNA, instructions were given to complete the assigned annotations and to put the values into PhageNotes.

Future Plans:

Further introductions to more databases will be given in order to create the best annotation for Napoleon B.

Rationale:

After a well-informed lesson on the database of NCBI, notes were put into the notes box in order to store information for the gene.

Tools:

• Personal Laptop
• DNA Master
• Phage Elesar

Results:

• After a further explanation on how temperate phages choose between lysogenic and lytic cycles, a lesson was given over the notes that were to be put into the note box to enable for the gene to be put into storage.
• Notes for gene 1 were completed.
• A QTM 5 was given for practice in which information from the BLAST results was used for gene 46 and 47 with the help of the current annotation guidelines.

Gene 46

Gene 47

Conclusion:

From the lesson over the notes that were to be put into the note box, some practice was done over gene 46 and 47.

Future Plans:

In the future, we will continue annotating gene 1 and using this information to understand DNA Master.

Rationale:

To further understand the functions of DNA Master and how to utilize the tools in order to fully annotate a gene

Tools:

• Personal laptop
• DNA Master
• Phage Elesar

Results:

• DNA Master was opened.
• Phage Elesar was downloaded and annotated.
• From gene 1, notes were put into the note box on DNA Master for storage.
• Some practice was also done with the QTM 4 to better understand parts of NCBI which provide information such as the alignment window, description, max score. total score, query cover, e value, identity, and accession.

Conclusion:

From the QTM 4, the information that was given was used for practice in order to better understand the data for future annotations. Gene 1 was used in order to understand the notes that were to be put into the notes box.

Future Plans:

Now able to understand the data, information will be put into the notes section in DNA Master in order to label the gene.

Rationale:

The purpose of this lab was to further understand DNA Master and the information presented for the future.

Tools:

• Computational Device
• DNA Master program
• Elesar FASTA File

Results:

• After the presentation about Ebola was given, DNA Master has been opened and the phage Elesar FASTA file was uploaded.
• The file was then exported in order for the sequence to be created.
• After it was exported, it was auto-annotated from the option of genome then auto-annotate.
• After annotation, the information under Features was explained such as gap and overlap calculations and practice was done with gene 19.
• Then, the frames were explained and purpose for their different structures along with the purpose of six reading frames.
• The ORF’s were viewed and future tools were discussed.

Conclusion:

From this lab, DNA Master was explained into more detail such as which preferences were needed and which steps needed to be taken to view the open reading frames. Future plans were explained and which tools were needed to accomplish those tasks. We also practiced gap and overlap calculations in order for future research.  Along with the calculations, the 5′ to 3′ for each base pair was explained and they were supposed to be read.

Future Work:

In the next lab, DNA Master will further be explained and actual annotation will begin.

Rationale:

From a computational device, changes were made to the system to accommodate and gain the necessary information for the lab. Also, from the FASTA file with the phage Elesar, the genome of the phage was used for practice to understand the information processed on DNA Master.

Tools:

Computational Device

DNA Master

FASTA File of a phage

Results:

• After presentations, the preferences on DNA Master were changed to provide for easier access to the information needed for the lab. The preferences that were changed were the colors which labeled the different segments of information and some slight changes in the settings.
• After the preferences were changed, a FASTA file containing the genome of the phage “Elesar” was downloaded off of phagesdb.com which was used for practice.
• The file was unable to download under the file tab in finder; however, it did work when moved to the desktop.
• After the file was selected and exported, auto-annotated was selected in which allowed for the open reading frames to be identified.
• In order for open reading frames to be evaluated further, a feature allowing for the 5′ to 3′ was selected.
• Through these steps, more practice was completed in order to become familiar with the program.

Conclusions:

After DNA Master was downloaded, the preferences were changed to allow for better management of the program. Although some difficulty was encountered when the FASTA file was downloaded, it worked when moved to the desktop then moved to the program. After the file was placed in the program, instructions were placed on how to work the program and where certain options were located. Some practice was done with the phage “Elesar” to become familiar with the program for future use.

Future Works:

Next class, the information that is present will be taught and will allow us to understand what is being displayed in order to annotate the phage “NapoleonB.”

Rationale: To calculate the titer and determine whether or not a new plate has to be made to achieve a high titer. If not, flood each plate.

Procedure:

• The titer of each plate with phages present was calculated and determined if the titer was high enough to be considered with pure phage.
• The plate containing 1X concentration and 2X concentration were flooded with 8 mL of phage buffer than placed on the shaking incubator.

Results and Analysis:

Conclusion:

The titer of 1X concentration and 2X concentration were calculated and flooded.

Future Plans:

DNA Extraction

Rationale: Due to the negative results of the plaque assays, modifications were made to the procedure to achieve a high titer.

Procedure:

• Three sets of dilutions to the 10^-4 were made by adding 90 microliters to each tube.
• For one set of dilutions, 7 microliters of the 10^0 lysate were used then diluted.
• For the second set, 14 microliters of the lysate were diluted to the 10^-4
• For the third set, 30 microliters were used.
• Using the 10^-4 dilution from each set, three plaque assays were made.
• Each dilution was combined with 0.5 mL of Arthrobacter and left alone for 10 minutes for infection.
• Then, three sets of cultures were made using 2.0 mL of LB Broth and 22.5 microliters of calcium chloride.
• The infected Arthrobacters of each dilution with a culture then top agar was added and plated.

Results and Analysis:

Conclusion:

Three concentrations of an original lysate were made with dilutions to the 10^-4. The first concentration was 7 microliters of lysate then diluted. The 2X concentration consisted of 14 microliters and 3X concentration consisted of 30 microliters. Using the 10^-4 of each concentration, plaque assays were made using the lysate, Arthrobacter, LB Broth, calcium chloride, and top agar.

Future Plans:

Calculate the titer of each plate then flood in order to get more lysate for DNA extraction

Rationale: In order to move to DNA extraction, 10 mL of lysate of pure DNA is needed. Due to a shortage of lysate, plaque assays were created in order to flood and obtain the lysate of pure DNA.

Procedure:

• After contamination prevention measures were taken, 125 microliters of lysate was added to 0.5 mL of Arthrobacter and allowed for infection for 10 minutes.
• In another test tube, 2.0 mL of LB broth and 22.5 microliters of calcium chloride were added together.
• After the 10 minutes, the lysate and Arthrobacter was added to the LB Broth and calcium chloride.
• Then, 2.5 mL of 1X Top Agar was added to each test tube then quickly added to the plate. To solidify, the plate was left alone for 15 minutes.

Results and Analysis:

First Phage

Head: 52 nm

Tail: 180 nm

Second Phage

Head: 47 nm

Tail: 106 nm

Conclusion:

Due to the inability to reach the minimum amount of lysate needed for DNA extraction, plaque assays were made to be used for flooding. In order to do this, plaque assays were made using 125 microliters of lysate was added with 5 mL of Arthrobacter then left alone to infect. For the culture, LB broth and calcium chloride were combined together. Then, the infected Arthrobacter was added to the culture. After, the top agar was added then plated.

Future Plans:

If phages are present, the titer will be calculated and will be used to determine if it is a high titer. If there are no phages present, plaque assays will be done again.