SIMULATING THE EFFECT OF HYPEROXYGENATION AND CIPROFLOXACIN ON PSEUDOMONAS AERUGINOSA POPULATIONS

I would say my interests are STEM-oriented; my favorite subjects are math and science. I was looking for a project that could be computer-oriented, and this was an ideal path. Pseudomonas aeruginosa is a bacteria that is often present in the biofilm-ridden lungs of cystic fibrosis patients. The antibiotic-inhibiting properties of biofilm combined with the bacteria's quick adaptation and resistance to antibiotics means that Pseudomonas infections are typically chronic affairs. The ability to determine how outside factors influence its resistance to antibiotics (such as Ciprofloxacin) would be a valuable asset. In this case, I want to know how introducing excessive amounts of oxygen would affect bacterial growth and antibiotic effectiveness. As such, I am trying to use results from fellow MARC student Anya to make computer simulations that predict exactly that. This project takes a population dynamics approach instead of focusing on changes in individual cells, hereby provided a big-picture view.

Edward '18: News

WAY OVERDUE UPDATE

February 15, 2018

So it's been three months to the day since my last blog post... but don't worry, it's justified! I've done a lot!

Right before winter break (well, technically during), I spent around 13 hours in the lab collecting data for a growth curve. This meant measuring the optical density of a plate of bacteria every 20 minutes, which ideally would give me a nice curve where I can see the bacteria populations start growing, grow for a while, and then slow down. I used two 96-well plates, which each had one row of blank wells on top for a control, and the other 7 rows had bacteria. One of the plates was shaken continuously, the other wasn't and both were shaken for five seconds prior to each measurement.

And it worked! Now I have around 168 individual growth curves, which I'm been playing with to make more manageable. I've linked a graph of one of my rows: https://imgur.com/a/xGgho

Now I just need to figure out how to digest this data and implement it into my program. When in doubt, do more curve fits!

DATA COLLECTION INBOUND

November 15, 2017

Monday I had another meeting with Dr. Hall of Dominican University, this time about how I want to structure the math behind my simulation. I've figured out the user interface tools by now, and so I've spent the past week restructuring my previous iteration to better separate the UI and math. Now, I can change one without having to tweak the other, which makes things a lot easier. If I want another variable, then I can add it it one spot and have it show up everywhere - previously, changing the math meant I needed to manually redo various other aspects.

Now that I can change my formulas easily, it makes sense that I should focus on beefing those up. Dr. Hall suggested I ought to figure out what factors might influence bacteria growth rates: amount of growth medium, total number of bacteria, oxygen concentration or flow rate, and anything else. To do so, I'll need to do some "calibration curves:" testing that shows how the actual bacteria performs, which I can then use as a basis for my computer model. I've got a few papers that should help me with the lab-to-math process; time to get reading!

PROMISING PYTHON PROGRESS

October 16, 2017

Since the beginning of the year, my knowledge of programming has expanded significantly. So far, nearly everything has been self taught. Instead of being a disadvantage, I find it's actually a lot of fun. I'll come up with an idea, think through what needs to be done, and try to figure out how to do it. It's just one big, self-directed puzzle!

So far, much of my time has been spent trying not to program mathematical functions but the user interface. This has included working with a utility called Tkinter to implement an output log, entry fields, buttons, check boxes, and something still in progress: a graph. The last item has been giving me some trouble, but I've been making working through the issues and think the finished product will be pretty neat. While the UI might not be essential at the moment, I figure at some point down the line I'll need to come up with something so I might as well figure it out as I go. As it stands, it looks good and is functional, so that's neat.

SCIENCE SYMPOSIUM RUNDOWN

October 5, 2017

I realized that I forgot to cover my finished science symposium on the blog! The short version is that it went well: all except one of the plates did what they were expected to do, while the outlier was resistant to ciprofloxacin when, in theory, it shouldn't have been. I'm not sure whether that's due to it mutating on the fly or bad lab practices on my part, or some other factor I haven't considered. None the less, it's significant. I then made a simple program that would say whether bacteria would grow or not based on various conditions.

SECOND EDITION

September 29, 2017

A lot has happened since the last blog post. After school restarted, I did a literature review and met again with Dr. Hall to discuss what I ought to do going forward. I then made another research proposal, this time with an updated goal, timeline, and resources.

As of late, I have been piecing together the framework of another program in Python. The user interface is done with Tkinter and is mostly done (except for the graph), as shown below.

Clicking "Run Simulation" will calculate bacterial populations based on the values entered by the user to the left, and will display the result in the output log. The graph does not yet function, but that's something I'd like to implement soon.

As soon as I get the framework working smoothly, I want to do some research as to how outside factors can influence bacterial growth, and whether there is an upper limit to said growth. That way, I can update the math to make it more indicative of an actual bacterial population.

STARTING THE PROCESS

May 1, 2017

Today, I was finally able to start lab work. I introduced bacterial colonies to the antibiotic resistance plasmid, heat-shocked them so they would accept it, put them on plates, and set them to incubate. There were 5 plates:

growth medium, no plasmid
growth medium & antibiotic, no plasmid
growth medium & antibiotic, with plasmid
growth medium, antibiotic, and plasmid activator, no plasmid
growth medium, antibiotic, and plasmid activator, with plasmid

The next class, I examined them with a U.V. light to determine which had picked up the plasmid and which had not. The only one that was glowing was the one that should have, with the plasmid and activator. I then prepared new plates, this time with a different selection:

growth medium, no plasmid
growth medium & antibiotic, with plasmid
growth medium, antibiotic, and plasmid activator, with plasmid
growth medium, antibiotic, and plasmid activator, with plasmid
growth medium, antibiotic, and plasmid activator, with plasmid

These also worked as they should, and the three with plasmid and activator all glowed. From here, I might be able to make a computational model showing the development the probability of developing resistance.

EXPERIMENTAL DESIGN

April 23, 2017

The next step in my Science Symposium project is to finalize the procedure. While I have most of it laid out, there are a couple steps where I'm still not clear. Examples include how exactly I will be taking measurements, and the timing of those measurements.

To aid in this, I will be attending a Sterile Technique and Biohazard training along with the other students whose projects involve bacteria. That way, I can iron out any kinks before they become actual issues during the project. I will also be speaking with my teachers about issues specific to my project.

CHANGE OF FOCUS

April 13, 2017

For the upcoming Science Symposium, I'm going to be shifting my focus a little bit. I've decided that I will run some preliminary trials with E.coli using BABEC pGLO kits. This will work better in the short term since the school already has kits available, and requires minimal experimental design. Everything is pre-planned and there is the added bonus of the antibiotic-resistant bacteria glowing under UV light. Going through the process will enable me to better understand the concepts I plan to simulate, and will also help me to comprehend some of the factors and potential issues.

MEETING WITH THE EXPERTS

March 23, 2017

On Tuesday 3/14, I met with Dr. Randall Hall from Dominican University and Shawn Cole from Marin Academy. I learned more about their backgrounds: Dr. Hall specializes in computational chemistry and biology, while Shawn did a lot of computational work in graduate school. We discussed directions going forward, and decided to focus on populations of bacteria, instead of the changes an individual organism makes. Dr. Hall has software that is partially applicable, so I am looking into what I can do with that to make it more relevant to the task at hand.

Please feel free to contact me with any questions or thoughts.

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