Poster Review and Drafting 2/25/18

Rationale

The rationale behind these procedures is to ensure that poster creations for Scholar’s day follow the correct format and that basic poster requirements are understood.

Tools/Procedure

1. Previously created posters were presented to the class in order to communicate poster requirements
2. The class was separated into small groups for collaboration
3. A basic poster layout was created for the QTM to demonstrate an understanding of basic poster requirements

Results

The above poster layout was designed by Rachel and I and was approved as an acceptable layout.

Conclusion

Lab period today was used mainly as an instructional period, so there aren’t any scientific conclusions to draw from what I learned. I can, however, say that I have an understanding of basic poster requirements so I should be able to make one in the future.

Future Plans

In the future, I will be working with Rachel and then with larger groups to create a poster for Scholar’s day.

Reviewing Annotation and Poster Review 2/20/18

Rationale

The rationale behind these procedures is to ensure that all annotations on NapoleonB are correct and the abstract is prepared for Scholar’s Day.

Tools/Procedure

1. Several annotations were examed as a class to ensure correctness
2. Genes were reexamined using:
   • NCBI was used to BLAST amino acid sequences against a large database of recorded sequences
   • Starterator and genemark were used to determine if the genes ought to be altered to include more or fewer base pairs
3. The information gathered was used to verify the gene calls that were previously made
4. Poster requirements were reviewed to increase understanding

Results

Upon reviewing annotations a gene between genes 66 and 67 was called. It was discovered to have NKF and I will finish it annotations by Monday lab (see image above).

Conclusion

There is not a lot of information from which to draw conclusions, but it is interesting to see that there a gene in between 66 and 67 because there was no coding potential shown on genemark.

Future Plans

In the future, I will use the annotations of NapoleonB to be able to research my own questions and hopefully add in someway to knowledge about phage.

Reviewing Annotations and Group Abstract Writing 2/18/19

Rationale

The rationale behind these procedures is to ensure that all annotations on NapoleonB are correct and that abstracts are prepared for Scholar’s Day.

Tools/Procedure

1. Several annotations were marked as potentially erroneous and were reexamined
2. Genes were reexamined using:
   • NCBI was used to BLAST amino acid sequences against a large database of recorded sequences
   • Starterator and genemark were used to determine if the genes ought to be altered to include more or fewer base pairs
3. The information gathered was used to verify the gene calls that were previously made
4. Then in order to have an abstract prepared several lab members collaborated to create an abstract

Results

This image shows the unchanged gene 66 after it was reviewed

The abstract that was created by group collaboration is written below:

“Bacteriophage phage represent a widely diverse and understudied population of microorganisms. In order to further aid in the effort to understand the mechanics behind bacteriophages, the 2018-2019 Phage Hunter class of Baylor University isolated Arthrobacter phages from soil samples collected throughout Waco, Texas. The samples were washed and the resulting lysates were plated to test for phage presence. Phage presence was indicated by a series of plaque assays, spot tests, PCR and gel electrophoresis. Once phage presence was confirmed, the process of purification and amplification was used to achieve a high titer lysate which was then archived for DNA extraction. DNA was extracted from NapoleonB, sequenced, and late annotated using DNAMaster and other bioinformatics tools. In bench research, NapoleonB was found to constantly display two distinct plaque morphologies and appeared to be a Myoviridae in the AM phage cluster. In silico research revealed that NapoleonB contains a linear double-stranded DNA genome with approximately 97 genes composed of 57846 base pairs. Future research can be used to determine the exact mechanics behind NapoleonB’s many functions.”

Conclusion

There is not a lot of information from which to draw conclusions, but I am able to assert that gene 66 was called correctly.

Future Plans

In the future, I will use the annotations of NapoleonB to be able to research my own questions and hopefully add in someway to knowledge about phage.

The Forgotten Cure 1 2/18/19

In the first 4 chapters of The Forgotten Cure we learn about the early discoveries of phage, phage therapy, and phage biology.  Blog about which ideas or observations presented in the first 4 chapters surprised or shocked you concerning the process of scientific discovery? You may use these examples or comment on your own ideas.

1. Describe the role that locusts, dysentery and war had in the discovery of bacteriophage.
   • d’Herelle was very interested in infectious disease but he was poorly educated. As he began his research career one of his first few tasks was pest management and fermentation. As part of these tasks, d’Herelle experimented on locusts by infecting them with bacteria. From these experiments, d’Herelle made bacteria cultures, and it was there that he noticed that some cultures had clear circles in them. While d’Herelle did not investigate this phenomenon right away, it was one of the first recorded instances of bacteriophage plaques, and he would later return to continue his research. During the war research into how to control disease became more important because many soldiers were dying of disease, this meant d’Herelle had more support for his research and there was a greater sense of urgency.
   • Dysentery and war played a role in the discovery of bacteriophages because together they created a situation in which d’Herelle began to actually consider the plaques he had noticed before. During the war research into how to control disease became more important because many soldiers were dying of disease, this meant d’Herelle had more support for his research and there was a greater sense of urgency. When he was sent to study dysentery during the war, d’Herelle noticed the same clear areas as he had noticed when he studied locust. He then found that patients who were recovering seemed to show signs of these plaques while the ones who died did not. This led him to purify and research bacteriophage.
2. Discuss the characteristics of d’Herelle that led him to be a successful scientist. How did he compare to Georgi Eliava?  What happened to the Eliava’s?
   • d’Herelle became a successful scientist because he was stubborn and had a genuine passion for what he did. While he was abrasive and argumentative, his being in the right place and the right time and being willing to really study his observations meant he was able to advance his scientific cause. Eliava, by contrast, was far more personable, while he was equally dedicated to scientific research, he was better educated and less abrasive, which allowed him to be successful in a different way.
   • However, because of Eliava’s connections to wealth and foreigners he and his wife faced trouble under communist rule. There were some ill feelings between Eliava and Beria and this eventually led to the Eliava’s being arrested by the Soviet secret police. They were shot at some point during the communist purge of intellectuals, wealthy people, and all other foreign interests. Meaning that d’Herelle outlived his student.
3. Discuss the influence war and politics had on the spread of phage therapy.
   • War, helped spread phage therapy because it made new disease treatment methods a necessity. World War one in part helped spur on the discovery of bacteriophages, and their therapeutic uses made phage therapy research highly valuable, which allowed it to spread. Phages meant that soldiers could survive what they might not have otherwise, and because world wars one and two had massive body counts, ways to save lives became a huge priority.
   • In addition, political competition such as that, that existed between the USSR and the capitalist world meant that a large emphasis was placed on scientific advancement as a way to compete or catch up to other nations. For phage therapy, the soviet union was choosing to compete in many areas including phage research and d’Herelle was able to visit and continue his research there, after he left Yale.
4. What are some of the reasons that the spread of phage therapy failed?
   • Phage therapy failed for a variety of reasons, but the first is that after Eliava dies, d’Herelle stopped promoting his work and stopped doing new research. He instead chose to live a more comfortable life and without his enthusiasm phage research lost a valuable advocate. In addition, there was a lot of conflicting information about phages in scientific research. Doctors weren’t doing double-blind trials or following other necessary scientific procedures so results from experiments with phage were often inconclusive or contradictory. Also, phage were a lot more temperamental and so it was harder to reproduce even positive results. This led to JAMA publishing increasingly less favorable reviews of phage therapy that began to dissuade physicians from using them. Finally, with the introduction of medicines like sulfa drugs, phage therapy became less popular as researchers began to focus on developing new antibiotics with less severe side effects. This research led to the discovering of penicillin which eventually became widely used. While there was still some phage research going on, it began to fizzle out and was eventually widely abandoned as a medical treatment.
5. How did the physicists Delbruck and Luria end up as part of the Phage Group? What contributions did they make to phage biology?  Why did phage biology die out in the 70’s?
   • Both Delbruck and Luria ended up as part of the Phage Group by trying to find ways to combine physics and biology.  Luria was inspired by a paper Delbruck had coauthored, so he began to research bacteriophages. Delbruck also became interested in phages and when both of them chose to flee to America to escape Nazism they met and began to work together. They laid the foundation for Phage Group as a collection of researches using phage to study genes and their contributions grew from there. Phage group discovered that phage had complex structures including the phage tails that typify phage shape today. In addition, they proved that DNA was the genetic material when they showed that it was phage DNA and not proteins that entered the bacteria cell. Phage Group made the study of bacteriophage legitimate and cleared the way for many more scientific discoveries.
   • However, phage biology began to die out. In the 70s phage biology died out because many scientists were intrested in understanding functions and once the phage was understtod they felt there was little else to do. Also phage group had chosen to only research 7 phage species limitng the scope of research that could actually be done.

Annotation Practice and Elesar Phage Notes 2/6/2019

Rationale

The rationale behind these procedures is to learn how to add annotations to phage notes and how to use phamerator and starterator.

Tools/Procedure

1. DNA Master was opened and the previous Elsar file was loaded
2. Learned how to use phamerator
3. Previous annotations were checked and added to phage notes
4. Phages DB was used to compare gene start calls to starterator results

Results

Gene 10 – SSC:7514 – 7813, CP:No Didn’t want overlap, SCS:Both, ST:NI, BLAST-Start:No significant NCBI BLAST alignments, No significant PhagesDB BLAST alignments, Gap:2bp gap, LO:NA, RBS:Kibbler7 and Karlin Medium 2.299 -4.08 Yes, F:NKF, SIF-BLAST:NKF, SIF-HHPred:NKF

Gene 11 – SSC:7829 – 8230, CP:No no start codon avaiable to cover the coding potential, SCS:Both, ST:SS, BLAST-Start:Aligns with Arthrobacter phage Ryan gpNA NCBI BLAST q1:s1 0.95 2E-70, Aligns with Phage Ryan gp12 PhagesDB BLAST q1:s1 0.96 2E-67, Gap:15bp gap, LO:Yes, RBS:Kibbler7 and Karlin Medium 2.565 -3.515 No, F:head-to-tail adapter, SIF-BLAST:head-to-tail adapter Supported by NCBI BLAST Phage Ryan gp12 AYN59006.1 0.95 2E-70, head-to-tail adapter Supported by PhagesDB BLAST Ryan gp12 MH834627 0.96 2E-67, , SIF-HHPred:NKF, SIF-Syn:head-to-tail adapter, upstream gene is NFK, downstream gene is Head-to-tail stopper, just like in phage Ryan

Conclusion

These results are very similar to the results of last lab with the exception of the starterator information. The results further support that gene 11 is a head-to-tail adapter and that gene 10 is an orpham that codes for a hypothetical protein not common in other sequenced DNA. Because this was just a learning experience not much else can be concluded from these results.

Future Plans

In the future, I will use what I learned how to do in this procedure when I am analyzing the genome from Napoleon B. Annotating in phage notes will be extremely helpful because they give me correctly formatted annotations that can be copied into DNA master to prevent any mistakes. I will also begin to use tools such as starerator when making calls about genes from Napoleon B.

BLAST Practice and Annotation Notes 1/30/2019

Rationale

The rationale behind these procedures is to learn how to annotate genes in DNA master by looking at BLAST results and using them to add information to the annotation notes.

Tools/Procedure

1. DNA Master was opened and the previous Elsar file was loaded
2. The annotation notes for the following fields – SSC: CP: SCS: BLAST-Start: Gap: LO: RBS: – were completed for gene 1
3. In order to complete the annotation notes the start codon number for gene one was adjusted from 84 to 45
4. Genes 10 and 11 were BLAST-ed and the annotation notes for the following fields – SSC: CP: SCS: BLAST-Start: Gap: LO: RBS: – were completed

Results

Gene 1 – SSC: 45,353 CP: yes SCS: both-cs BLAST-Start: no significant BLAST alignments Gap: first LO: yes RBS: Kibler7, Karlin Medium, 1.222, -6.751, no

Gene 10 – SSC:7514,7813 CP: yes SCS: both BLAST-Start: no significant BLAST alignments Gap: 15 bp LO: yes RBS: Kibler7, Karlin Medium, 2.299, -4.080, yes

Gene 11 – SSC: 7829, 8230 CP: Yes SCS: Both BLAST-Start: Aligns with Arthrobacter phage Ryan, NA, NCBI, q1:s1, 95%, 2e-70 Gap: 15 LO: yes RBS: Kibler7, Karlin Medium, 2.565, -3.515, no

Conclusion

These results show the completed structural annotation notes for genes 1, 10, and 11. Of these genes, only gene 11 has significant BLAST results, meaning that gene 11 has a sequence of DNA very similar to other DNA found in bacteriophage. Genes 1 and 10, however, do not have significant BLAST results and therefore are not highly conserved among known genomes. This could mean that hypothetical genes 1 and 10 are not real genes, or it could mean that they are unique and could code for more unique protiens.

Future plans

In the future, I will use what I learned how to do in this procedure when I am analyzing the genome from Napoleon B. I will perform auto-annotations as well as many other forms of testing on that genome in the lab periods to come. I will likely BLAST all of the genes in Napoleon B and compare those results to genemark and the auto-annotation results in order to asses that all genes are in fact genes and to fully annotate the genome. I will also be sure to fill in all of the basic information that is required in the annotation notes to make scientific inquiry easier in the future.

BLAST Practice 1/28/2019

Rationale

The rationale behind these procedures is to learn how to annotate genes in DNA master by looking at BLAST results and using them to add information to the annotation notes.

Tools/Procedure

1. DNA Master was opened
2. A FASTA file was opened (File > Open> FastA Multiple Sequence File)
3. The contents of the file were exported (Export > Create Sequence from this Entry Only)
4. The contents of the file were Auto Annotated (Genome > Auto Annotate > click “yes”)
5. The reading frames were examined (Genome > Frames> click “ORFS” button)
6. Genes 1 and 2 were selected to BLAST (click on gene number > BLAST tab > Qblast against public database via NCBI server > Blast this gene)
7. Results from the BLAST were examined and used to start filling in the annotation notes

Results

The procedure detailed above was simply practice that yielded the above results. These results show what an auto-annotated genome would look like after being BLAST-ed before the annotation notes have been updated. These results allow one to asses whether or not a gene that was marked as a gene during auto-annotation is actually likely to be a gene and it gives a frame of reference for comparing the query DNA sequence to other DNA sequences.

Conclusion

The results of the practice BLAST can be seen above. These results allow one to compare the query DNA sequence to similar sequences of DNA as one means of verifying that a gene is a real gene, and as a way to help understand the function of the gene and potentially identify areas of conserved DNA in multiple genomes.

Future plans

In the future, I will use what I learned how to do in this procedure when I am analyzing the genome from Napoleon B. I will perform auto-annotations as well as many other forms of testing on that genome in the lab periods to come. I will likely BLAST all of the genes in Napoleon B and compare those results to genemark and the auto-annotation results in order to asses that all genes are in fact genes and to fully annotate the genome.

DNA Master Annotation Practice 1/23/2019

Rationale

The rationale behind these procedures is to learn how to annotate genes in DNA master while also adding several features to the auto-annotation sequence.

Tools/Procedure

1. Open DNA Master
2. Open File> Preferences > Local Settings > New Feature > add the following code:
   SSC: CP: SCS: ST: BLAST-Start: Gap: LO: RBS: F: SIF-BLAST:
   SIF-HHPred: SIF-Syn
3. Open a FASTA file (File > Open> FastA Multiple Sequence File)
4. Export contents of the file (Export . Create Sequence from this Entry Only)
5. Auto Annotate contents of the file (Genome > Auto Annotate > click “yes”)
6. Examine reading frames (Genome > Frames> click “ORFS” button)
7. Compare gene locations paying attention to gaps and overlap

Results

The procedure detailed above was simply practice that yielded the above results. These results show what an auto-annotated genome would look like for future reference in later annotations. They also show the 6 possible reading frames on which genes can be found. The genes shown highlighted in green signify genes read in the forward direction while genes highlighted in red show reverse genes. These results allow me to compare gaps and overlaps between genes to determine in the auto-annotation was accurate in determining genes in the genome.

Conclusion

The results of the practice auto-annotation can be seen above. An annotated genome that can be reviewed by a human. By examining the reading frames one can determine if the genes marked in the auto-annotation are likely to be legitimate coding strands for genes or not. While there were no tough calls to make in today’s lab, genes 25 and 22 look unlikely to be legitimate and this will need to be further examined.

Future plans

In the future, I will use what I learned how to do in this procedure when I am analyzing the genome from Napoleon B. I will perform auto-annotations as well as many other forms of testing on that genome in the lab periods to come. I will also likey study genes 25 and 22 to ensure that my initial reaction to their legitimacy is accurate.

 11/28/18 Spot Titer Test, TEM, and DNA Extraction Part 1

Objective:

The goal of this procedure is to assist Lucy P. in getting a large amount of high titer lysate. This is achieved by webbing and then flooding plates. This procedure will detail the process of calculating the titers of the lysate C (the result of webbing plates with lysate 1), creating a TEM grid, running a TEM, and creating a DNA pellet as part of DNA extraction.

The overarching question this test seeks to address is: Is the presence of phage determined by species of oak tree from which soil was collected?

In other words, are specific oak tree species more likely to have Arthrobacter bacteria phages in the soil surrounding them?

The question specific to my lab table is: Is the difference in the presence of phage between live oaks and red oaks on Baylor’s campus?

As a group, we hope to expand our question to include more species as we gather data so that we can better address our overarching question and we will look at our metadata to examine whether or not there are other factors that may determine phage presence.

Procedures and Protocols:

Materials for an Aseptic zone:

• CiDecon
• 70% Ethanol
• Ethanol Burner

Materials for a Serial Dilution:

• Phage Buffer
• Microcentrifuge Tubes
• Vortex Machine
• Pipette

Materials for Lysate Filtering:

• Topfilter
• 50 ml conical
• Flooded webbed plates

Materials for Phage Precipitation:

• High Titer Lysate
• 50 ml conical vial
• Nuclease mix
• Phage Pericpitate solution

Materials for TEM Grid:

• High titer lysate
• 400 mesh copper grid
• Grid box
• TEM forceps
• DI water
• Uranyl Acetate
• Gloves
• p20 micropipettor
• Parafilm

Materials for a Spot Test:

• .5 ml Arthrobacter
• incubator
• Pipette
• Test tube stand
• 50 ml tubes
• Culture tube
• LB Broth
• 2X TA
• 1M Calcium Chloride
• Agar plate
• Serological pipette

In order to complete the procedure, an aseptic zone was created.

1. CiDecon was applied to the lab table with a squeeze bottle and wiped away with a paper towel
2. 70% Ethanol was also applied with a squeeze bottle, spread with a paper towel, and allow to evaporate
3. An ethanol burner was light in order to use the rising heat from the flame to form the aseptic zone
   

Lysate C was collected and filtered

1. The lysate from the flooded plates was poured into a top filter
2. The lysate was filtered into a 50 ml conical

The Phage Precipitation was performed.

1. 10 ml of lysate C were transferred to a conical vial
2. 40 µL of Nuclease mix were added and the vial was inverted several times to mix
3. 4 ml of phage precipitate were added
4. The tube was placed in the incubator for 30 minutes
5. Then the tube was left at room temperature for ~40 minutes
6. The tube was then centrifuged at 10,000g for 20 minutes *Note: the tubes got locked in the centrifuge machine overnight before being centrifuged for 20 minutes*
7. The supernatant was poured into the sink and the pellet was put in the freezer until next lab

While phage precipitation was performed a TEM grid was created.

1. 20 µL of Lysate, 20 µL of DI water, 20 µL of DI water, and ~20 µL uranyl acetate were placed on a strip of parfilm as shown below:
2. TEM forceps were used to place a copper grid shiny side down in the lysate for 5 minutes
3. Forceps were then used to transfer the grid to each sample of DI water for 2.5 minutes each
4. Finally, the grid was transferred to the uranyl acetate for a minute
5. Excess moisture was wicked away with filtered paper and the grid was loaded into the TEM for imaging

Then serial dilutions were performed on lysate C.

1. Seven levels of dilution were created for each lysate (called lysate 1 and 2): 10^-1, 10^-2, 10^-3, 10^-4, 10^-5, 10^-6, 10^-7
2. Seven microcentrifuge tubes were filled with 90 µL of phage buffer
3. 10 µL of lysate C (10^0) were transferred to one of the 10^-1 vials
4. The tube was vortexed to mix
5. 10 µL of the solution was taken the 10^-1 tube and transferred to the tube labeled 10^-2
6. The tube was vortexed to mix and this procedure of dilutions was repeated through the 10^-7 dilution for lysate C

Then the spot titer test on the new lysates was performed.

1. One agar plates was labeled as shown:
   
2. Agar was prepared according to the following recipe (makes two plates):
3. 4.5 ml of the agar was transferred to the labeled plate
4. The plate was swirled and set aside to allow the agar to solidify
5. When agar was solidified, 10 µL of phage buffer as well as 10^-3, 10^-4, 10^-5, 10^-6, and 10^-7 dilutions for each lysate were transferred to their appropriate spot on the plate

Results:

Update: As can be seen from the image above, our spot test failed. We know our lysate is a high titer, so it seems likely we inverted the plate too quickly so the lower concentration dilutions didn’t have enough time to absorb onto the agar.

In addition, the results of our TEM imagining can be seen. In our group, 2 distinct types of phage were found, as detailed above.

Analysis:

The idea behind these procedures is to learn more about our phage as a whole. From these procedures were were able to learn about our phage morphology and extract DNA for further testing. This is the final step needed to ensure we sucessful have a phage that we can add to the phage database.

Future:

We will finish DNA extraction next lab and we will archive.

11/16/18 Spot Titer Test

Objective:

The goal of this procedure is to assist Lucy P. in getting a large amount of high titer lysate. This is achieved by webbing and then flooding plates. This procedure will detail the process of calculating the titers of the lysates created during the last few labs by doing a spot titer test.

The overarching question this test seeks to address is: Is the presence of phage determined by species of oak tree from which soil was collected?

In other words, are specific oak tree species more likely to have Arthrobacter bacteria phages in the soil surrounding them?

The question specific to my lab table is: Is the difference in the presence of phage between live oaks and red oaks on Baylor’s campus?

As a group, we hope to expand our question to include more species as we gather data so that we can better address our overarching question and we will look at our metadata to examine whether or not there are other factors that may determine phage presence.

Procedures and Protocols:

Materials for an Aseptic zone:

• CiDecon
• 70% Ethanol
• Ethanol Burner

Materials for a Serial Dilution:

• Phage Buffer
• Microcentrifuge Tubes
• Vortex Machine
• Pipette

Materials for Lysate Filtering:

• Syringe filter
• 50 ml conical
• Flooded webbed plates

Materials for a Spot Test:

• .5 ml Arthrobacter
• incubator
• Pipette
• Test tube stand
• 50 ml tubes
• Culture tube
• LB Broth
• 2X TA
• 1M Calcium Chloride
• Agar plate
• Serological pipette

In order to complete the procedure, an aseptic zone was created.

1. CiDecon was applied to the lab table with a squeeze bottle and wiped away with a paper towel
2. 70% Ethanol was also applied with a squeeze bottle, spread with a paper towel, and allow to evaporate
3. An ethanol burner was light in order to use the rising heat from the flame to form the aseptic zone
   

Lysate 2 was collected and filtered

1. A syringe was used suction lysate off of the flooded plates
2. The lysate was filtered into a 50 ml conical

Then serial dilutions were performed on each lysate.

1. Five levels of dilution were created for each lysate (called lysate 1 and 2): 10^-1, 10^-2, 10^-3, 10^-4, 10^-5
2. Ten microcentrifuge tubes were filled with 90 µL of phage buffer
3. 10 µL of the previously created lysate 1 (10^0) were transferred to one of the 10^-1 vials
4. The tube was vortexed to mix
5. 10 µL of the solution was taken the 10^-1 tube and transferred to the tube labeled 10^-2
6. The tube was vortexed to mix and this procedure of dilutions was repeated through the 10^-5 dilution for lysate 1
7. 10 µL of the previously created lysate 2 (10^0) were transferred into the other 10^-1 vial
8. The tube was vortexed to mix
9. 10 µL of the solution was taken the 10^-1 tube and transferred to the tube labeled 10^-2
10. The tube was vortexed to mix and this procedure of dilutions was repeated through the 10^-5 dilution for lysate 2

Then the spot titer test on the new lysates was performed.

1. One agar plates was labeled as shown:
   
2. Agar was prepared according to the following recipe (makes one plate):
3. 4.5 ml of the agar was transferred to the labeled plate
4. The plate was swirled and set aside to allow the agar to solidify
5. When agar was solidified, 10 µL of phage buffer as well as 10^-3, 10^-4, and 10^-5 dilutions for each lysate were transferred to their appropriate spot on the plate

Results:

As can  be seen from the image above, our lysate spots traveled. When calculating it appeared that lysate 1 had a 10^8 titer and lysate two had a 10^7 titer.

Analysis:

The idea behind the procedures as a whole is to enable us to create larger quantities of high titer lysate. In theory, this can be done after a plaque has been purified by creating webbed plates that can be flooded with phage buffer. The resulting mixture then holds many phage, and the titer can be tested with a plaque assay or spot test. We have been amplifying the titer of our lysates, so this spot test should reveal a high titer lysate.

Future:

Because neither lysate produced quite a high enough titer, Lucy and I will need to web more plates with the hopes of creating a higher titer lysate to use TEM.