Uncovering Phage StrongArm: Part 3… The Final Chapter

Woah, it’s been a few months since I was last here so let me summarise what you lucky people have all missed out on. Can you believe it’s been 145 days, or 20 weeks and 5 days or 4 months and 23 days since you guys have heard from me?! Not to be exact or anything 😅 Anyways… the phage “hunt” program has FINISHED!! I know it’s sad to hear but all good things come to an end.

Sadly, I didn’t get to annotate StrongArm [1], instead he was in the very safe hands of Dr Heather Hendrickson [2]. Myself and another phriend Tyler [3] ended up helping our phriend Bailey by annotating her phage Bazzle [4]. We enjoyed our time spent with Bazzle and making a video to show people how to identify and trim tRNA’s.

The annotation process took a “tedious” amount of time, but it was still fun while it lasted. It involved using different programs called PECAAN [5], Phamerator [6] and DNA Master [7]. I know those names may be all complicated and mean nothing to you right now so let me delve into it for you guys a little bit to explain what each are used for.

PECAAN is an important program which allows you to go through each gene and choose a start and stop site and the function it may have… if it even has one. As well as being able to add and delete genes. This process used BLAST, HHPRED, GeneMark and Starterator to help figure out all of these little details, as well as using ARAGORN and tRNAscan-SE 2.0 to identify and trim tRNA’s. However, I’m not going to bore you with all the details of each one, otherwise you’ll be asleep in no time.

Figure 1. Image

Another important program used was DNA Master which helps give you more information for allowing you to choose specific start sites in closer detail by getting a representation of what each gene in the genome looks like as shown in the figure below. It also allows you to see all the coding potential and if there’s any coding regions where a new gene may be able to be added, which makes you feel awesome when you discover one!!

Figure 2. Image of an open reading frame analysis of PatrickStar from DNA Master’s quick user guide on the SEA-PHAGES website. The genes are shown here by the red and green highlights. Red is showing a reverse gene, while green shows a forward gene.

The last important program involved in annotating a phage genome is Phamerator. This allows anyone to get a good visual representation of each gene in a phages genome and how the genes differ to other phage from either different of same clusters. There is more differences between phage from different clusters than phage within the same cluster. This allows us to see the similarities and differences between phages. StrongArm is a part of the A1 cluster, while Bazzle is a part of the L2 cluster.

Figure 3. Image from Phamerator showing comparison between the genomes of Bazzle, Archie and BigCheese. It shows how similar both phage are, with only a few genes that differ in length and location. Many of the genes also belong to the same pham group, shown by the colour of each gene (e.g. gene 12 for both Bazzle, Archie and BigCheese are a part of the same pham (37389) shown by the orange colouring).

I hope this and my last two posts have helped you all get a better understanding and feel for what the whole phage “hunt” experience was like. This year has been a whirlwind of emotions from losing my first phage to finding another one. It’s been a great adventure getting to learn new things, with lots of laughs along the way. It’s sad to say goodbye as so much time and effort has been put into this that it’s like a chunk of me will be missing, but I wouldn’t trade this experience for the world.

Goodbye my phage phamily 🙂

References

1. PhagesDB: the actinobacteriophage database. (2019). Mycobacterium phage StrongArm. Retrieved from https://phagesdb.org/phages/StrongArm/

2. Massey University. (2019). Dr Heather Hendrickson. Retrieved from School of Natural and Computational Sciences – biology cluster: https://www.massey.ac.nz/massey/learning/colleges/college-of-sciences/about/natural-sciences/inms-staff/inms-biology-staff/inms-biology-staff_home.cfm?stref=953250

3. PhagesDB: the actinobacteriophage database. (2019). Mycobacterium phage Gavin. Retrieved from https://phagesdb.org/phages/Gavin/

4. PhagesDB: the actinobacteriophage database. (2019). Mycobacterium phage Bazzle. Retrieved from https://phagesdb.org/phages/Bazzle/

5. Rinehart, C. A., Gaffney, B. L., Smith, J. R., & Wood, J. D. (2016). PECAAN: User Guide [PDF file]. Retrieved from SEA-PHAGES: https://seaphages.org/media/docs/PECAAN_User_Guide_Dec7_2016.pdf

6. Cresawn, S. G., Hatfull, G. F., Bogel, M., Mavrich, T., Gauthier, C., & HHMI SEA-PHAGES. (2006-2018). Phamerator. Retrieved from http://phamerator.org

7. Lawrance, J. (2007). DNA Master. Retrieved from http://cobamide2.bio.pitt.edu/

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Finalizing the chaos and the fun

Throughout this year we learnt and achieved a lot but also spilled a lot of blood, sweat, tears, and phage buffer (tell a lie, no blood was actually spilled but it sounded cool).

So what did we actually achieve this year?

We cumulatively collected and processed [1] hundreds of environmental samples and eventually found our own bacteriophages. I’m not overexagerating this time either, I alone collected 92 different environmental samples. As a class, all 6 of us had our own baby phages to characterize and care for.

We named and became very attached to our baby phages. Check out my phage, Golddigger in one of my previous blogs “Now I ain’t saying she’s a Golddigger…oh wait…”.

We characterized our phages!
We did this via a range of techniques as explained in our lab books [1];
We completed a gel electrophoresis (Image one A) [2] to identify if our phages were different.
We looked at our phages under an electron microscope (Image one B) and used this to calculate their capsid size (for Golddigger this was 109.68nm) and tail length (for Golddigger this was 48.39nm) as well as identified their morphology (Golddigger is syphoviridae).
We analysed our phages plaque morphology and size,
I even had time to analyse the heat tolerance of Golddigger. As seen in Image two, the amount of plaque forming units per ml (pfu/ml) drastically decreases from 70c onwards. Identifying this is important for any future research on Golddigger, for example the use of Golddigger in phage therapy (the importance of which is explained by [3]).

Image Two
Logarithmic graph showing the estimated pfu/ml of Golddigger at different treatment temperatures in °C for 10 minutes and then diluted and plated via spot testing.

We isolated our phages DNA and 3 of the 6 phages collected were sequenced over the semester break and annotated using semester two which turned out to be a more complex then expected.

We all made it to the end of a really hard year!

So what have I taken away from this experience?

I learnt a lot of valuable laboratory (which I’m proudly showing off in image three), writing and, research skills which will be useful for my future endeavors.
These writing skills also taught me that different scientific disciplines require different formats and that apparently my brain is more wired to psychological scientific writing rather then biological scientific writing.

Image Three
Showing off my lab skills. Photo curtsy of Heather Hendrickson.

I am less technology illiterate then I thought! (I’m still pretty bad though…)

I’ve accepted that failing and mistakes is a necessary part of science and it’s okay and that there will always be smart people around you to help you pick up the pieces both literally and metaphorically (sorry about spilling my phage buffer Jarred).

So as I am approaching the final hours before everything in this paper is due and I rush to get everything finished, I thought it would be nice to reflect on the year that I have just had, the knowledge I have gained and the friends that I have made so,

  1. Hendrickson, H. (2019). 246202 Bacteriophage Discovery and Genomics. Course and assessment guide.
  2. https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcr-electrophoresis/a/gel-electrophoresis
  3. Aldayel, M. F. (2019). Biocontrol strategies of antibiotic-resistant, highly pathogenic bacteria and fungi with potential bioterrorism risks: Bacteriophage in focus. Journal of King Saud University – Science. https://doi-org.ezproxy.massey.ac.nz/10.1016/j.jksus.2019.08.002

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What a year it has been

The year is coming to an end and it’s time to reflect. In my first blog I wrote about the resilience I needed while collecting samples. In my second blog I talked about the highs and lows of ‘bonding’ and being proud of my Bazzle. In my third blog I detailed the sequencing information of Bazzle’s DNA and the excitement of going into annotating. 

Would I have taken this course if I knew what I now know? Hell yeah! Taking Bacteriophage Discovery and Genomics has clarified the direction of my career path. Being in the lab is where I belong. This course gave me the taste of what biological researching in a lab requires. During the year I discovered my own capabilities, weaknesses and desires. 

One of my main highlights was going to Auckland University to see the electron microscopy (EM) (1). I felt very privileged as a second-year student to be able to view my bacteriophage using the machine. I loved every second of seeing everyone’s excitement as each phage was loaded and viewed on the EM. 

Image taken by Nikki Freed at Auckland University

As well having amazing experiences, through this course I was able to improve techniques within the lab that I struggled to do beforehand. One example of this that comes to mind is gel electrophoresis (2). In previous courses my hands would shake so much that I would miss the well in the gel or didn’t put the pipette tip far enough that the dye would spill out of the wells. When we were required to do a gel electrophoresis this year, I was very nervous that I would make similar mistakes however, this was the first time that I did everything perfectly. 

Image taken by Heather Hendrickson of me completing the gel electrophoresis

Writing a yearlong manuscript was one of my main challenges I faced this year. Trying to format it in a scientific way was new to me. However, I’m glad that I still got to experience what writing one is like. I believe that this knowledge will benefit me in postgrad when I will be required to do something similar. 

Another challenge I faced was the annotating process, I found it tricky to get my head around and understand all the different components of it. Although, I did quickly begin to understand bits and pieces. This process although tricky, was still very much exciting to learn something I have never done or thought about before. It was also intriguing to me to be able to see all the genes and functions within my own phage. 

On reflection, I am forever grateful for Heather and her knowledge, teaching style and approachability that made this course one to remember and one to help further my career in this amazing field. I am also very grateful of the amazingly talented humans that I became friends with this year. I do not regret taking this course at all. It is one of my best decisions I have ever made to date. Personally, I have never felt prouder of myself then I have this year. This class has empowered me and enabled me to grow more as an undergrad student and as a young scientist.

Thank you to all that have been a part of this journey or have read these blog series of my journey. If you are another student taking this course after 2019 then my advice to you is soak up every moment of this experience and enjoy the excitement of discovering your very own phage. 

References:

  1. UMASS medical school. (n.d). What is Electron Microscopy? Retrieved from https://www.umassmed.edu/cemf/whatisem/ 
  2. Nature education. (2014). Gel electrophoresis. Retrieved from https://www.nature.com/scitable/definition/gel-electrophoresis-286/

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Dermatologists hate her

A large majority of people are no strangers to pimples- especially when one, two or a few crop up right before an important outing. Even I continue to have my fair share of having hotspots, and not just limited to the face. Acne can be triggered by a number of complex factors, such as diet, stress, hormone imbalances and bad face touching habits. But also more likely due to bacteria, specifically Propionibacterium acnes (Williams, Dellavalle, & Garner, 2012). P.acnes thrives off sebum secreted from sebaceous glands found predominantly on the face as well on the upper chest and back (Endly & Miller, 2017). For many of us, dabbling in an array of remedies ranging from clay masks to chemicals and medicines is not uncommon, each with variable outcomes. It would come to a time where you can no longer care and have simply given up.

And so I had a shower thought- so there’s bacteria living in my pores that’s giving me grief on my face. And phages are viruses that infect bacteria. Oh? OH. If there’s phage therapy for bacterial infections, wouldn’t it also be effective in stopping acne once and for all and we can all have baby skin?

It turns out that this shower thought isn’t the first of its kind!

Multiple attempts had previously been made by various researchers to isolate P. acnes by collecting skin scrapings from volunteers with clear skin and those suffering from acne. Jończyk-Matysiak et al. (2017) had found that virulence of P.acnes causing inflammatory responses leading to acne was dependent on the presence of certain strains. Analysis by Liu et al. (2015) showed human skin was commonly only colonised by  a single P. acnes strain. Furthermore phages showing active infection of P. acnes was more frequently isolated from volunteers with clear skin, suggesting the regulatory role phages play in human skin (Liu et al., 2015).  Other isolation projects were also conducted by (Brown, Petrovski, Dyson, Seviour, & Tucci, 2016), and a separate study by (Marinelli et al., 2012) had yielded 11 P. acnes phages from healthy skin, all showing high degrees of homogeneity. Characteristics of tested phages, such as having a lytic lifecycle and absence of genes associated with lysogeny, showed a certain degree of promise in using phage therapy for treatment of acne.

This is a facial product for clearing skin of acne. Ingredient of interest is active bacteriophage targeting Cutibacterium acnes, which is also known as P. acnes. Though I could not find further information about it.
Image from https://twitter.com/markowenmartin/status/1145029207078031360.

Brown et al. (2016) had even trialled a topical cream by infusing Cetamacrogol cream with phage particles with a concentration of 2.5×108 pfu/ml per gram. The cream could remain active for 90 days in storage environments without light at 4°C, where it was effective in lysing P. acnes cells in lawn cultures (Brown et al., 2016). This could potentially be the antibiotic alternative to acne treatments albeit only trialled in laboratory settings.

Nonetheless, this brings joy to my face knowing research for potential acne cures have not been forgotten and are underway. So in the meantime, I will have to make do with homemade banana masks and keeping picking fingers at bay.

References
Brown, T. L., Petrovski, S., Dyson, Z. A., Seviour, R., & Tucci, J. (2016). The Formulation of Bacteriophage in a Semi Solid Preparation for Control of Propionibacterium acnes Growth. PLOS ONE, 11(3), e0151184. doi:10.1371/journal.pone.0151184

Endly, D. C., & Miller, R. A. (2017). Oily Skin: A review of Treatment Options. The Journal of clinical and aesthetic dermatology, 10(8), 49-55. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/28979664

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605215/

Jończyk-Matysiak, E., Weber-Dąbrowska, B., Żaczek, M., Międzybrodzki, R., Letkiewicz, S., Łusiak-Szelchowska, M., & Górski, A. (2017). Prospects of Phage Application in the Treatment of Acne Caused by Propionibacterium acnes. Frontiers in Microbiology, 8. doi:10.3389/fmicb.2017.00164

Liu, J., Yan, R., Zhong, Q., Ngo, S., Bangayan, N. J., Nguyen, L., . . . Li, H. (2015). The diversity and host interactions of Propionibacterium acnes bacteriophages on human skin. The ISME Journal, 9(9), 2078-2093. doi:10.1038/ismej.2015.47

Marinelli, L. J., Fitz-Gibbon, S., Hayes, C., Bowman, C., Inkeles, M., Loncaric, A., . . . Modlin, R. L. (2012). Propionibacterium acnes Bacteriophages Display Limited Genetic Diversity and Broad Killing Activity against Bacterial Skin Isolates. mBio, 3(5), e00279-00212. doi:10.1128/mBio.00279-12

Williams, H. C., Dellavalle, R. P., & Garner, S. (2012). Acne vulgaris. The Lancet, 379(9813), 361-372. doi:https://doi.org/10.1016/S0140-6736(11)60321-8

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It’s a bird, It’s a plane … Nope it’s a tRNA!

DNA annotation is the final step of successfully capturing a phage and really gets into the knitty gritty. Through the use of PECAAN [1] , Phamarator [2] and DNA master [3] software I was able to get to the bottom of which genes coded for particular functions in Phage Bazzle [4]!

Bazzle EM Picture

Phage Bazzle (https://phagesdb.org/phages/Bazzle/)

After feeling like an annotation expert, I was blind sided by a bizarre large gap between genes, which can be seen when looking at the start sites of genes of Bazzle in PECAAN. When further investigating, DNA master revealed these to be tRNA’s. TRNA’s are transfer adaptor molecules that serve as the link between mRNA and the amino acid sequence during translation [5]. Within the sequence tRNA’s don’t appear as normal coding in the genome, therefore the software identifies that these are rogue. They are often easy to spot by a signature gene gap as which puzzled me since they appear in clusters, multiple tRNA’s in the same region. The can also be seen on Pham Maps of PECAAN represented as +’s. Bazzle hoarded 11 cheeky tRNA’s that all required special annotation.

PECAAN showing tRNA of Bazzle as +

TRNA annotation is making sure these molecules are the correct structure, containing the right bases on their acceptor stem loop. This loop must have CCA bases at the 3′ as well as 7bp in total. Errors after transcription can alter these bases so we’ve gotta be prepared to give them a trim (even if it’s a mullet, yikes).

Image result for tRNA loop
tRNA with acceptor loop containing CCA at 3′ end (http://www.bx.psu.edu/~ross/workmg/TranslationCh14.htm)

Thankfully gene annotation is ahead of the game and I simply had to run the scripts of tRNA through Aragorn [6] and tRNAScanSE [7] which develop two options for which they think the tRNA should be trimmed. The best one is which matches the conditions above, this is then confirmed in PECAAN and saved along with the rest of the annotation, BOOM done.

And just like that a year of phaging has finished! I will forever be grateful for the experience and confidence this course has given me, as well as the passion it has sparked for microbiology. The future is looking exciting!

Bibliography

  1. PECAAN annotation software https://seaphages.org/media/docs/PECAAN_User_Guide_Dec7_2016.pdf
  2. Phamerator annotation software https://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-12-395
  3. DNA Master Software https://phagesdb.org/DNAMaster/
  4. Phage Bazzle https://phagesdb.org/phages/Bazzle/
  5. tRNA definition https://www.nature.com/scitable/definition/trna-transfer-rna-256/
  6. Aragorn Software https://www.ncbi.nlm.nih.gov/pmc/articles/PMC373265/
  7. tRNAScanSE Software https://www.ncbi.nlm.nih.gov/pmc/articles/PMC146525/

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The Final Characterisation of the rare Bacteria found while Phaging!

I have learnt so much about the microbial world this year and my passion for it has continued to bloom. It’s amazing where academia can take you when you find a subject that sparks such interest. Phage hunt (1) has been an exciting adventure. Not only do you learn an incredible amount about the world of phage, but you gain real life laboratory experience and annotation skills that you would be hard pressed to find in other second year papers.

PhaginSelfie

Me, happily attempting to find a phage during Phage Hunt.

In a parallel manner, I have also learnt a lot about the world of bacteria; the two go hand in hand. I have previously written about the predatory bacteria (2) I thought I had discovered. Check out that blog to read about those cool little warriors! I am very lucky that the team at Massey University gets just as excited as me about a discovery like this, and we could carry out further testing!

The first, easiest step, was to stain the bacteria (3) with crystal violet and iodine. This is a routine protocol (4) when working with bacteria and characterises the bacteria based on the structure of their cell wall (5). Gram negative bacteria go pink or red under staining, because they have a thinner cell wall and the dye is not retained. Gram positive bacteria go purple; the thicker cell wall, and the peptidoglycan in it, holds onto the purple in the stain and does not get decolourised by the iodine. My bacteria went pink – confirming it is gram negative.

Predatory bacteria tend to be gram-negative (6), but at ~0.75 micrometers by ~3 micrometers (7), they are larger than a phage . I wondered how it would be possible for a bacterium to get through the miniscule filters we use to separate the phage out. This became clear when I saw the small, rod-like shape. It must have gone through sideways and then multiplied from the lucky few that got through, causing the plaque like shape on the plate.

Transmision-electron-micrograph-of-Pseudomonas-mendocina-MC2-Magnification-40-000

Transmission electron micrograph (14) of Pseudomonas mendocina MC2. Magnification, 40 000 ×. 

The next step was to isolate the bacterial DNA using the Wizard Genomic Prep Kit (8) and sequence it on a Nanopore MinION (9) machine. A MinION works by running a piece of DNA through a protein nanopore in the bottom of the machine. As each base is pushed through the pore, an electrical current reads the sequence and displays it in real time. The MinION is fast and relatively effective, plus it’s a student friendly sequencing tool. From the data we discovered the bacteria was Psuedomonas Mendocina (10).

nanoporeMinion

A Nanopore MinION sequencer (9)

P. mendocina is a dirt-dwelling bacterium and rarely infects humans, but when it does, the effects can be serious. It was first discovered in 1970 (11), in Mendoza, Argentina. There have been 8 cases of human infection recorded in the scientific literature (12), none fatal. It works by entering the body through a pre-existing infection, and then travels through the bloodstream until it reaches the heart valves, where the infection tends to settle. I was very excited to finally know what my bacterium was, especially considering the uncommon nature of P. mendocina. The world of molecular biosciences is so interesting, and to have the ability to discover and identify a bacterium like this one was an experience that has inspired and motivated me to continue on this career path. I can’t wait to see what other cool things science brings me in the future!!

References:

  1. The Bacteriophage Discovery and Genomics paper homepage https://www.massey.ac.nz/massey/learning/programme-course/course.cfm?course_code=246202
  2. Blog: The Mystery Bacteria https://wordpress.com/read/blogs/67175144/posts/2098
  3. Gram Stain Technique https://vlab.amrita.edu/?sub=3&brch=73&sim=208&cnt=2
  4. Methods of Classifying and Identifying Microorganisms https://courses.lumenlearning.com/boundless-microbiology/chapter/methods-of-classifying-and-identifying-microorganisms/
  5. Gram Positive vs. Gram Negative Bacteria https://www.thoughtco.com/gram-positive-gram-negative-bacteria-4174239
  6. Gram-Negative Versus Gram-Positive (Actinomycete) Nonobligate Bacterial Predators of Bacteria in Soil https://www.ncbi.nlm.nih.gov/pmc/articles/PMC239120/
  7. Psuedomonas Microscopic Appearance https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/Pseudomonas.htm
  8. Wizard® Genomic DNA Purification Kit Protocol https://worldwide.promega.com/resources/protocols/technical-manuals/0/wizard-genomic-dna-purification-kit-protocol/
  9. Oxford Nanopore MinION https://nanoporetech.com/products/minion
  10. Pseudomonas mendocina native valve infective endocarditis: a case report https://www.semanticscholar.org/paper/Pseudomonas-mendocina-native-valve-infective-a-case-Rapsinski-Makadia/1fcc2c391457b5c26fc9effd5a7869efb3e46a85
  11. Taxonomy of the Aerobic Pseudomonads: the Properties of the Pseudomonas stutzeri Group https://www.microbiologyresearch.org/content/journal/micro/10.1099/00221287-60-2-215
  12. Pseudomonas mendocina native valve infective endocarditis: a case report https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5048412/
  13. Gram Staining: Principle, Procedure and Results https://microbeonline.com/gram-staining-principle-procedure-results/
  14. Transmission electron micrograph of P. mendocina https://www.researchgate.net/figure/Transmision-electron-micrograph-of-Pseudomonas-mendocina-MC2-Magnification-40-000_fig1_237865575
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Beginning to understand my baby Bazzle

Luckily for you my avid followers, you get an extra blog written by me. I slept in and missed a phage hunt lab last semester and in order for it not to affect my grades this is what I had to do.  On another note I am very excited to announce that Bazzle was one of three phages in the 2019 phage hunt crew to be sequenced.   

The results from the restriction digest gel electrophoresis gave me an idea on how Bazzle differed from the other phages in the phage hunt, however the results from the sequencing indefinitely confirms how much different Bazzle is. All the sequencing information for Bazzle can be found on the phageDB1. For starters the genome length was a whooping 76,734 bp long which is an estimated 2,000 more than the two other sequenced phages, strongarm and squee. Bazzle is also found to be in cluster L with 53 other discovered phages around the world and subcluster L2 shared with 23 other phages.  According to phageDB, a phage in the L cluster is typically temperate. Temperate phages are phages that produce lysogens and can choose between the lytic and lysogenic pathways2.  

Bazzle had a GC content of 58.7% and an overhang length of 10 bases containing the sequence TCGATCAGCC. Research suggests that there is a correlation between the genome length and the GC content, the longer the genome length, the higher the GC content. Because my genome length is quite long this could be the reason why my GC content is 58.7%. However when comparing my results to other phages this reasoning is quickly thrown out the window. Phages with higher GC content tended to have shorter genome lengths. As you can see science is still learning about the size, shape and uses for phages. I am very excited to be part of this new frontier and delighted that Bazzle is unique.  

From the results retrieved from the sequencing, I can’t decipher a lot of what it means in terms of my phage information. This is why I am very excited for this semester where annotation will take place. I cannot wait to see what all this information means and what new information I will discover about Bazzle. 

Stay tuned for my next blog which will probably be about what I find during annotation. 

References:

1. https://phagesdb.org/phages/Bazzle/

2. Phage consultants. (n.d.). Temperate phages.Retrieved from http://www.phageconsultants.com/temperate-phages,16,pl.html

3. Almpanis, A., Swain, M., Gatherer, D., & McEwan, N. (2018). Correlation between bacterial G+ C content, genome size and the G+ C content of associated plasmids and bacteriophages. Microbial genomics, 4(4).

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Now I ain’t saying she’s a Golddigger…..oh wait….

Ladies and Gentlemen, let me introduce you to Golddigger!

Image one: Electron microscope image of Phage Golddigger.

Now I’m sure your asking, Why is your phage named Golddigger? If you’ve read my previous blog “The side of scientific research that they don’t tell you about…” then you’ll understand the issues and stress that we had during this years phage hunt and so Golddigger is rather fitting as this phage has demanded alot of my time and energy!

Golddigger was found in compost in the Hobsonville Point community garden in Auckland, New Zealand (as seen on this map; Golddigger was found where the yellow star point is. https://www.google.com/maps/d/u/0/edit?mid=17CSRYFl1B39D-lElJ2cR3x2m14ygn3CW&ll=-36.76670858962872%2C174.49151200000006&z=11 ). Samples 81-86 were all found here. I kept sample 81 which is Golddigger, sample 83 was adopted to Winnie Zeng, Courtney Armstrong and, Tyler Porteous and sample 86 was adopted to Jamie Le Roux.
We were very fortunate that in the community garden there was three very large compost bins (the bins were approximately 1.4m x 1.4m x 1.2m in length, width and height respectively) and I was able to collect 6 samples (81-86), two from each bin (one from the front and one from the back), from here which all came back with positive plaques as seen in appendix three.

As you can see in image one above, Golddigger is a siphoviridae type bacteriophage who’s tail is approximately 109.68nm long and the capsid has a diameter of approximately 48.39nm.
The full calculations for these sizes are in appendix one.

Golddigger’s morphology presents as large clear plaques which on average are 4.875mm in diameter (as seen in image two below), however the plaque sizes ranged from 3.75mm to 6mm. Golddigger and another phage (squee found by Jamie which can be seen in appendix three, plate 86) showed very similar plaque morphologies and so a gel electrophoresis was completed and is detailed below.

As you can see in image three, Golddiggers DNA isn’t easily cut by restriction enzymes. Upon analysis, the only enzymes that cut Golddigger’s DNA were ClaI, HaeIII and possibly SalI as it shows a slightly lower band (it traveled 2mm further then the uncut DNA did) then all of the other restriction enzymes.
From this analysis, it appears that Golddigger’s DNA is larger then 1kb because the uncut DNA sits 3mm higher then the 1kb marker. (3)

By completing a gel electrophoresis analysis, we were able to identify that Golddigger is distinctively different from all of the other phages found in the class. This was important because I found all of the phages used by the class, except for phage bazzle which was found by Bailey in a different location.
You can see all of the other gel electrophoresis results in appendix two.

So, despite all of the stress and tears that we had at the start of the semester with our imposter host bacteria, check out appendix four if you want to learn about that, I’m the proud mum of phage Golddigger who is beautiful and unique in it’s own special way!

Appendices

Appendix one
Calculations showing how Golddiggers tail length and capsid diameter were worked out. Calculations are based on the calculations in reference two.

Tail length = 109.68nm
(scaled size bar / Measured size bar) = (unknown scaled size / measured size)
(50nm / 31mm) = (unknown scaled size / 68mm)
unknown scaled size = (50nm x 68mm) / 31mm
= 109.68 nm

Capsid diameter = 48.39 nm
*same calculation as above except the measured size is 30mm, therefore,
Unknown scaled size = (50nm x 30mm) / 31mm
= 48.39 nm

Appendix two
All six gel electrophoresis images of all of the phages found in this years class. As you can see, Golddigger shows a distinctly different banding pattern to all of the other phages found.

Appendix three
Plates showing the positive plaques of samples 81-86.

Appendix four
These two plates below were made by our laboratory technician, Jarred, who suspected that the host that we were using for the first half of the semester had become contaminated somehow. He spot tested our phages and a control phage (FancyPants, which is in green in the bottom right hand section) on the old host (left) and the a new sample of the correct host (right). As you can see, FancyPants didn’t infect the old host and so from this he concluded that the reason why we were not finding any phages is because the old host that we were using had become contaminated by something else at some point.

References.

  1. Map showing where samples were taken from during phage hunt. The Yellow star is where Golddigger was found.
    https://www.google.com/maps/d/u/0/edit?mid=17CSRYFl1B39D-lElJ2cR3x2m14ygn3CW&ll=-36.76670858962872%2C174.49151200000006&z=11
  2. Calculations based on the calculations detailed in the 2019 Course and assessment guide, 2019 by Dr Heather Hendrickson.
    Protocol 8.1a, page 68.
  3. Gel electrophoresis set up and analysed using the phage hunt course and assessment guide 2019 by Dr Heather Hendrickson.
    Protocol 10, pages 77-87.
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Uncovering phage StrongArm: Part 2…

Let’s see, where did I leave off? Well… If I can remember clearly and correct me if I’m wrong, I was struggling over the loss of my original phage (1) StrongArm. I must admit the first couple of weeks were hard. I had to start from scratch and get used to this new and hopefully improved phage StrongArm. It’s like a 2.0 version where it is meant to be all superior and more suitable. I could never replace my original StrongArm though, but I had to move on with my research and this was the only way I could. I ended up renaming my original phage to StrongLeg, so that I could keep the name StrongArm for the phage I will be using.

Plate that I adopted off as well as two fellow Phage Hunters. I adopted one of the small lytic (clear) plaques circled with the blue pen.

This new phage I adopted from a fellow Phage Hunter, who gave myself and two others a plate that had undergone one round of serial dilutions (2) and displayed plaques (3) with different morphologies (3) (as shown above). I picked the small lytic (1) (clear) plaques, while the second person picked the large lytic (clear) plaques and the third person picked the lysogenic (1), turbid (cloudy) plaques.

Luckily the new phage lived up to the 2.0 description and it was smooth sailing from here on out. As I approached the same stage I was up to before my original phage stopped working, I was nervous and apprehensive to see what my fate will be this time… Phew! All was well. Nothing disastrous happened this time…

One of the plates I used to collect my high titer lysate

This meant I could now go onto the exciting next stage in this process which is DNA EXTRACTION (4)! This is what everything has lead up to. DNA is needed for the significant step of DNA sequencing (5) which is required to distinguish the differences and similarities between all the other bacteriophages found around the world which are entered into the database on PhagesDB (6). Only a selected couple from my Phage Hunt group will be chosen though, so fingers crossed!

Since my two other Phage Hunters and I got our phages from the same sample, the thought of them actually being the same phage was always at the back of my mind. This thought was confirmed through doing a gel electrophoresis (7) of a restriction enzyme digest (8) of my DNA, which showed that they were all the same phage. This was a tad disappointing as it would’ve been nice to each have our own phage, as it would’ve made it more interesting to see the differences between the appearances in our phage when looking at them under an electron microscope (EM) (9), (image shown below). Due to an EM costing over a $1 million its not exactly something each university would have just sitting around in a dark room. This meant we were off on a ROAD TRIP!

You could tell the day was going to be great when it started off jamming 6 people into a 5-seater car just to get to the bus station. Then you got to have your turn at using a $1 million equipment just to look at our phage, which are smaller than the wavelength of violet light (10) which is 400nm! (So of course we can’t see it with our naked eye). We had spent our whole semester working with our phage but we had never actually seen what they looked like, which is why it meant so much to us. You could feel the excitement and joy radiating off everyone. The look of awe on everyone’s faces as they got their first glimpse of their phage. Can you believe that at one stage I was looking at an image of my phage which was 110,000x magnified?! (Image shown above). That just shows you how little phages are, approximately 200 nanometers in size (11) to be exact!

Now its just a waiting game until the next semester starts back to see what phage got their DNA sequenced. From here onwards unfortunately there is no more practical lab work. Instead I’m trading the practical lab for a computer lab where I get to stare at a computer screen looking at A, T, G, C’s all day long analysing the data we will get given from our DNA sequencing. To check out my phage on the PhagesDB database click StrongArm (12).

References:

1. Kahn Academy. (n.d.). Bacteriophages. Retrieved from https://www.khanacademy.org/science/biology/biology-of-viruses/virus-biology/a/bacteriophages

2. Hatfull. G., Jacobs-Sera. D., Pope. W., Poxleitner. M., & Sivanathan. V. (2018). Protocol 6.2: Serial Dilutions. The Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science. Supported by Howard Hughes Medical Institute. Retrieved from https://seaphagesphagediscoveryguide.helpdocsonline.com/6-2-protocol

3. Griffiths. A., Miller. J., Suzuki. D., Lewontin. R., & Gelbart. W. (2000). Bacteriophage Genetics. An Introduction to Genetic Analysis. 7th edition. Retrieved from https://www.ncbi.nlm.nih.gov/books/NBK21824/

4. Hatfull. G., Jacobs-Sera. D., Pope. W., Poxleitner. M., & Sivanathan. V. (2018). Chapter 9: Extracting Phage DNA. The Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science. Supported by Howard Hughes Medical Institute. Retrieved from https://seaphagesphagediscoveryguide.helpdocsonline.com/9-0-overview

5. yourgenome. (2013). What is DNA sequencing? Retrieved from https://www.yourgenome.org/stories/what-is-dna-sequencing

6. PhagesDB. (n.d.) The Antibacterial database at PhagesDB.org. Retrieved from https://phagesdb.org/

7. Hatfull. G., Jacobs-Sera. D., Pope. W., Poxleitner. M., & Sivanathan. V. (2018). Chapter 10: Characterizing Phage Genomes by Restriction Enzyme Digests. The Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science.Supported by Howard Hughes Medical Institute. Retrieved from https://seaphagesphagediscoveryguide.helpdocsonline.com/10-0-overview

8. Hatfull. G., Jacobs-Sera. D., Pope. W., Poxleitner. M., & Sivanathan. V. (2018). Protocol 10.1: Setting Up Restriction Enzyme Digests. The Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science.Supported by Howard Hughes Medical Institute. Retrieved from https://seaphagesphagediscoveryguide.helpdocsonline.com/10-1-protocol

9. Tanabe. R. (2017). Electron microscope. New World Encyclopedia. Retrieved from //www.newworldencyclopedia.org/p/index.php?title=Electron_microscope&oldid=1006783

10. Science Learning Hub – Pokapū Akoranga Pūtaiao. (2012). Colours of light. Retrieved from https://www.sciencelearn.org.nz/resources/47-colours-of-light

11. Huang. C., & Huang. M. (2012). The Scale of the Universe. Retrieved from https://scaleofuniverse.com/

12. Armstrong. C. (2019). Mycobacterium phage StrongArm. The Antibacterial database at PhagesDB.org. Retrieved from https://phagesdb.org/phages/StrongArm/

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Horsin’ Around

Bacteriophage therapy has been under the radar of researches as the damning issue of antibiotic resistance begins to plaque the globe. As plan B antibiotics are being rendered useless, phage therapy has high hopes to save the crisis, but not only for humans. Phage research has been done on livestock in order to improve animal health and productivity. Of particular interest to me is the studies involving bacterial keratitis in horses, having owned a horse with keratitis I have first-hand experienced the difficult and expensive process of finding antibiotics that work.

Vet examining a horse with possible bacterial keratitis. Retrieved from https://equimanagement.com/articles/research-on-ulcerative-keratitis-in-horses

What is keratitis? Equine keratitis is an infection of the cornea often resulting in an ulcer which if left untreated can lead to blindness in the eye [1]. Racehorses are most prone to this disease due to their heads being in constant movement, increasing amount of eye surface area available. This creates a greater risk of the nasty bacteria creeping in [2]. It is also an issue in competition, such as in polo where horses who are blind, even if only one eye, are unable to compete. This disease can be identified by staining the cornea to see defects produced or production of an ulcer.

From L to R: Healthy horse eye without keratitis. Retrieved from https://www.horseandhound.co.uk/horse-care/5-incredible-facts-horses-eyes-636770 Horse eye with keratitis which has been stained. Retrieved from https://thehorse.com/130567/corneal-disease/ .

The biggest issue in treating keratitis is the emerging bacterial resistance. Getting the infection just once creates the horse to be more prone to contracting the bacteria again. This is often due to the environment the horse is in, being exposed to the same conditions increases the risk of it being in contact with the same bacteria [3]. As antibiotics are prescribed, they are becoming less and less effective, due to this overuse, leading to the question of what is next? Phage therapy was bought to the table and has produced promising results.

A study in Japan has shown this by the use of a model organism mouse [2]. They were able to incorporate the bacteria into the animal and successfully insert a bacteriophage to kill the strain before an ulcer could form. Their aim was to find an alternative to racehorse antibiotic washes which were used immediately after running to prevent infection to begin with. These washes were becoming ineffective and increasing bacterial resistance when applied so often. To begin with they followed a very similar protocol to find the bacteriophages we have done in Phage Hunt but instead of using Mycobacterium smegmatis [4] they used a Pseudomonas aeruginosa bacteria strain. Pseudomonas aeruginosa is a common rod-shaped bacterium found in both animals and humans [5]. They isolated [6] their samples from sewage material and were able to purify, amplify and view under an electron microscope just as I did with my phage Gavin.

Phage that were isolated from sewage material. Top phage are Podovirade and bottom phage are Myrovirdae. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988198/#B26

The resulting phage were identified to be of Myrovirdae and Podovirdae families, since Podovirade phage are more broad specific they decided to experiment on this phage for a greater chance of infecting the bacteria [7]. The mice were infected with the disease and phage samples were added, ensuring they were absorbed and not spilled out as tears. Results showed that the phage sample was effective until 3 hours after infection, the bacteria was successfully attacked by the phage and no ulcers or cornea damage was seen.

Although 3 hours is a very short period of time, for the purpose of preventing the infection in this racehorse situation it could be ideal to replace the antibiotic wash with a phage wash. The antibiotic washes are possibly not as effective due to not being well absorbed or retained when applied and it also helps to decrease antibiotic resistance.

My horse Millie who recovered from Bacterial Keratitis without going blind. Photo credit: Carole Pinkney

A small step in the right direction may be all it is but with more research phage therapy could be adapted for long term treatment of equine keratitis and other bacterial infections. Phage therapy, once forgotten could now be the answer to all our questions, including our newly discovered phage from Phage Hunt. Who knows, maybe one day my phage Gavin will be saving an eye or a life.

Bibliography

1. Nasisse, M.P. and Nelms, S. (1992) Equine Ulcerative Keratitis. Veterinary Clinics of North America – Equine Practice 8 (3), 537-555.

2. Furusawa, T. et al., Phage Therapy Is Effective in a Mouse Model of Bacterial Equine Keratitis, 2016, pp. 5332-5339.

3. Keller, R.L. and Hendrix, D.V.H., Bacterial isolates and antimicrobial susceptibilities in equine bacterial ulcerative keratitis (1993-2004), 2005, pp. 207-211.

4. Hatfull. G., J.-S.D., Pope. W., Poxleitner. M., & Sivanathan. V, The Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science Discovery Guide, 2018.

5. Cohen-Cymberknoh, M. et al. (2017) Clinical impact of Pseudomonas aeruginosa colonization in patients with Primary Ciliary Dyskinesia. Respiratory Medicine 131, 241-246.

6. Tanji, Y. et al., Spontaneous deletion of a 209-kilobase-pair fragment from the Escherichia coli genome occurs with acquisition of resistance to an assortment of infectious phages, 2008, pp. 4256-4263.

7. Jonczyk, E. et al., The influence of external factors on bacteriophages-review, 2011, pp. 191-200.

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