The Last of the Last (Legionella pneumophila Blog Week 12)

Wow oh wow this semester felt like a whirlwind! With classes, the S-STEM program and work it feels like the semester barely started yesterday yet here we are at finals week.  I just want to express my gratitude for all of the opportunities that this program has afforded me. Not only did I learn various lab techniques but I also had a taste of what research was actually like in a real life lab.  When we went to the ASU sequencing lab it really hit me that we were actually conducting the same research that you often hear of in the news.  Beyond the academic gains I have earned I also had the pleasure to meet some amazing people such as, my research team, the mentors and the people that are part of S-STEM. Thank you everyone and I hope that all of your academic and career aspirations come to fruition.

Estrella Mountain Community College Presentations (Legionella pneumophila Blog Week 11)

Hello everyone!  This week was presentations week at the Estrella Mountain Community College conference.  The conference was largely attended by people in the soft sciences so it was a little funny to see how technical all of our S-STEM posters where compared to what everyone else was presenting.  Despite the fact that we had really technical content we had a couple of our Phoenix College students win 2nd and 3rd place for poster presentations, congratulations to Beth and Oliver! Daisy and I also presented and I think it was a great experience to be able to talk to people about all of the work that we have been doing all semester long.  Here is a picture of Daisy, our poster and me!

Lab work on top of lab work (Legionella pneumophila Blog Week 10)

This week feels like we've been in the lab non-stop.  Tuesday our team finished filtrating 3 samples that we had not been able to filter last week because we ran out of autoclaved vacuum filtration glassware. Once we finished filtrating our samples we began DNA extraction from the filtered samples.  The only problem we had was that we were not using the usual DNA extraction kit that we had been using.  Instead we used a kit by Omega bio-tek. This kit used a pretty extensive protocol (roughly 30 steps) and it took our group a while to work through the extraction.  Wednesday we worked on our poster fine tuning what we think was wrong with it. I will post it here and would love to hear any feedback you guys may have on the poster.  Today also ran four Agarose Gels with the PCR product from our last PCR procedure in order to see if ASU's DNA Lab will be able to sequence these bands once we extract them.  We have our fingers crossed since we would really love to know through genomic sequencing that we found L. pneumophila in these samples.

Oh the Stress (Legionella pneumophila Blog Week 9)

Oh the stress, this week was pretty rough.  We had to get a great deal of work done in the lab plus we had to re-run gels and re-do some PCR on the samples we gathered because the sequencing lab at ASU was not able to sequence the samples that we extracted from the Agarose gels.  On Friday we also re-collected 8 samples in order to analyze them and see if they would test the same nearly a year later.

Despite the fact that we have had these setbacks I think that it has been a great learning week.  We got a chance to know what it is like to try to find results with an impending deadline (the Estrella Conference) looming over our heads.

Almost Through An Entire Cycle (Legionella pneumophila Blog Week 8)

I view our work in the detection of Legionella p. as a cycle.  First we must collect the samples, then we have to filter out the DNA, next is the extraction of the genetic material.  Once the genetic material is out, we amplify the DNA we want through PCR with primers specific to a gene in Legionella p. Once amplified we perform electrophoresis with a positive control and a molecular weight marker to screen for our suspect.  After all of this has been done we finally cut out the genetic material out of the gel, extract it and send it off for genomic sequencing at ASU. Then we start with gathering samples all over again.  This week we are supposed to send 4 of our samples off to ASU to get tested to see if we can absolutely identify the bacteria.  I can not wait to drop off our samples and see what we get.  Getting those results back would finally complete a cycle of research!

I'll post a picture of our group at ASU tomorrow when we go!
So here is the picture of the lab, I didn't want to take pictures of people so I tried to avoid them and this is all I could get.  

a

Even more PCR and Electrophoresis (Legionella pneumophila Blog Week 7)

Hello everyone, this is week 7 of our journey through the S-STEM internship!  This week for our Legionella p. project, we had to run PCR on 10 different samples.  We ran the PCR on Tuesday and today we ran the amplified DNA in the Agarose Gel. Unfortunately, I goofed on 4 samples that I loaded (I loaded the gel wells with 7 microliters [μL] of material instead of 12μL).  Our mentor, Robin Cotter, advised us that it would be wise to run the PCR again on these samples and do the electrophoresis again in order to verify our results.  The reasoning is that; if the Legionella p. bacteria are present but there was not enough genetic material in the 7μL then it may not be visible when looking at the gels.  As for the remaining 6 samples that did run and ran well, we will be cutting out the clearly visible bands and performing a DNA extraction process from the gels so that we can send the samples to ASU for genomic testing. Once we know the sequence of the nucleotides, we can compare that to the known sequence of Legionella p. and know for sure that the bacteria were present in the samples that we collected.

That is all for now! Above is a picture of me on my birthday with Daisy one of my group mates.

P.S. Today is my birthday!

1st Agarose Gel Pour/Run (Legionella pneumophila Blog Week 6)

Spring Break, short are thee
In your midst I wish
to be.  Once more free
from the petri dish

Yet here I am at,
close to the midnight.
Had I not laid flat
I'd be far from plight.

Hello everyone, that was my sadness from the end of spring break coming through.  I'll jump right into it since there is no better way to do work.  This week in the Lab our team ran PCR on nine samples some of which had been collected nearly 6 months ago.  I'm sure many of you are familiar with this process but after the PCR had been run. We poured 3 gels and ran our samples through 2 of them.  This was the first time I had ever poured the Agarose Gel and it being my first time it did not go well at all.  Luckily Daisy poured the other two and those were the ones we were actually able to use.  Below is an image of the water samples collected.  According to the banding size it appears as though we actually got a positive for the Legionella bacteria, on the first go around!  We didn't have enough time to scan the second gel but once I get the picture I will upload it onto here.  Coming back hasn't been so bad after all!
.

NanoDrop Pioneers! (Legionella pneumophila Blog Week 4)

Last Friday Josh showed our group (Daisy, Zaira and me) how to use the new NanoDrop machine! It was awesome to be the first ones to learn how to use it and actually get to run our samples through it.  Daisy even taught Bethany how to run her samples in the NanoDrop. 
     The NanoDrop measures the concentration of DNA in the any given sample.  It also has the ability to determine the purity of DNA in a tested sample by measuring the ratio of absorbance measured at wavelengths of 260nm and 280nm.  DNA tends to absorb at a wavelength of 260nm while protein, phenol and other contaminants absorb at a wavelength of 280nm (NanoDrop, 2007).  By comparing the two measurements, the NanoDrop is able to determine purity of DNA.  A ratio measurement for reasonably pure DNA would be around 1.8.  As you will see in the attached image, our samples do not display this type of purity.  The lowered purity is expected because our samples, as of now, do not contain purely DNA. The samples have other contaminants that affect this reading.  Daisy and Zaira have cut DNA samples out of agarose gels after PCR in the past and their results actually were aligned with what would be expected for pure DNA.  So we will get there soon!

NanoDrop, http://www.bio.davidson.edu/gcat/protocols/NanoDrop_tip.pdf

Doing Some Actual Work (Legionella pneumophila Blog Week 3)

I finally had the chance to do some actual work this week involving the collection of samples and working on extracting genetic material from the sample collected.  Oliver and I collected a sample of water from the cooling towers and we will be filtrating and extracting (if there is any) genetic material from our samples this week.  

Up until now I had been doing a great deal of background reading to familiarize myself with the bacteria, its significance and the methods that we would use in order to positively identify the presence of Legionella pneumophila.  

First up in the process was the actual collection of the sample.  Down below you'll see the location and a brief description of where the sample was taken from.  Once the sample was collected, it must undergo a filtration process in order to concentrate the contents that were in the sample into 5mL of water so that we can work with it better in the lab.  (FYI: Video is loud)

Once the samples have been concentrated the DNA (if there is any) of whatever was in that water is extracted from that sample using a Soil DNA Extraction Kit.  What this kit does is, take all of the genetic material that was in that sample and extract it from the organisms that were residing in the sample of water.  

This was my first time going through all of these steps and I think I was able to get through everything successfully, with the help of my teammates (Daisy and Zaira) of course!  I guess that's all for now, tomorrow we'll get a chance to use and learn how to use the new NanoDrop machine so I'm really excited about that.

Week 2 Legionella pnuemophila Research

Okay so here is an update on what is going on with this research project.  After laboring over the research proposal I have devised a possible route of exploration.  Much of the ground work has already been established by my partners in this project.  Their goal was to find out whether or not Legionella pneumophila was present in water systems around our school.  The samples they collected underwent a series of procedures that helped isolate the DNA of the bacteria as well as positively identifying L. pneumophila using genomic sequencing.

Using similar procedures to identify the presence of L. pneumophila I would be interested in seeing whether the bacteria has higher populations during the warmer months than the cooler months. What I am proposing to study is the concentration of DNA of the bacteria and compare this to the data that already exists.  The bacteria tends to like warm environments and is especially present in hot water systems.  Since Phoenix can get so hot I feel that they may present themselves in higher numbers by also accumulating in 'cold' water systems when temperatures reach the highs we all know and love.

However there are some obstacles that must be overcome in order for this experiment to be successful.  The biggest obstacle I foresee is being able to collect enough samples while it is still relatively cool.  The warm weather we felt was caused by a heat wave and hopefully I will be able to collect relevant samples when it cools down. If it cools down. 

Legionella Introduction

Legionella pnuemophila (Image source: theguardian.com)

 
Legionella pnuemophila pictured here, is a bacteria that can cause humans to develop acute pneumonia (also known as Legionnaire's disease or legionella pneumonia) 2-10 days after an individual breathes in a mist of water containing the bacteria.  In other words, when aeorosolized, the  inhaled bacteria can cause pneumonia.  Although pneumonia can oftentimes be easily treated at home with the disease ending after 2-3 weeks substantial complications can arise from individuals that have immune deficiencies such as the elderly.  Complications may also arise in individuals with diseases that affect the immune system such  as: cancer patients undergoing chemotherapy, people infected with AIDS among others.

 Presence of Legionella pnuemophila in water sources can pose health threats for high traffic areas, especially when there are numerous individuals in close contact with each other.  A college campus may be a perfect breeding ground for the propagation of Legionella pnuemophila and ultimately pneumonia in a given population.  For this reason knowing whether or not Legionella pnuemophila is present in the water and in biofilm around campus can help a campus implement ways to reduce the number of individuals that come into contact with this bacteria.

Knowing Legionella pnuemophila presence is the basis for the research that my team has been conducted in the past year and a half and at some point this research may help reduce the bacteria's population on campus or even eliminate it thus eliminating the potential for students, staff and faculty to develop pneumonia.