Semester 4- Week 13- The End.

Hello! And welcome to this, my final edition of this blog.

It has been an amazing ride these last 4 semesters in S-STEM. I have learned an incredible amount about science, but more importantly, I have made a number of great, soon-to-be-lifelong friends.

I will never forget the day I first interviewed for S-STEM. Anil Kapoor had recommended me for the program, but despite the endorsement of such a brilliant instructor, I was nervous. I felt out-of-place, and I was certain that I did not belong in such a prestigious program. My nervousness was only compounded when Matt Haberkorn inexplicably left the room during my interview, leaving me alone with Josh James. I thought for certain that I had screwed up somehow, and that Matt leaving the room was somehow a dismissal of my application.

Little did I know that Matt would turn out to be the kindest, gentlest person I would ever meet, and that he was incapable of that kind of insensitivity.

So there I was, face-to-face with Josh and his poker face. He asked me a question: "Who would you most like to have dinner with?" In a flash, I said. " I know a lot of people would say Jesus, but I want to meet .....". Josh's face was inscrutable, and again I thought I had flubbed the interview.

After what seemed like hours and a thousand other questions from Josh, Matt walked back into the room. Josh turned to him, and with that mischievous grin that I have seen a million times since, he spoke two words: "Jane Goodall." It was my answer to his question.

I then watched as these two guys silently communicated in the way that only two people who have known each other for years can do, with mouths silent but their eyes full of knowing; and I suddenly realized that I hadn't screwed up. In fact, I realized I had just met two people who understood me, for possibly the first time in my life.

In that moment I realized that I not only belonged, but that I had found a home. And the lab has been my home ever since- through good and bad, easy times and tough, even after I had my stroke and became a real pain in the ass, the Biosciences team has been there for me every step of the way. We have laughed together, expressed frustration, then laughed together some more-and through it all, I have felt love.

Thank you Josh, Matt, Dijana, Amanda, Cori, Ana, Kim, and Anil, along with all of the scholars that have befriended me along the way. My life has been forever changed for the better because of the home you have given me.

Live long, and prosper.

Paul.

Photo credit: Paul C. Selfie the day I got my shirt.

Semester 4-Week 12- Field Trip!

Hello all! Welcome back.

I am currently taking BIO 182, as apparently it was the only biology course I have NOT taken at Phoenix College, and it is required for my major. It is an interesting course, made even more so by the fact that I have an excellent instructor, John Schampel.

Recently, we were able to take a field trip to the Phoenix Mountain Preserve, aka the location of Piestewa Peak, and do some field work surveying dispersal patterns of ambrosia deltoidea. This lovely little shrub is commonly known as triangle-leaf bursage, and is an important Sonoran Desert native species that is related to sunflowers. See below.

 Photo credits: biohere.org. Bursage plants.

One of the benefits that this plant provides to the desert is its status as a microbiome. Bursage prefers to grow in open areas that receive a lot of sunshine, and therefore is one of the first plants to colonize hot, open spaces that are too hot for other plant seedlings. Its dense branch canopy also prevents herbivory. Bursage thus acts as a "nurse species" for other plant varieties. Over time, seeds from other species will germinate in the shade of a bursage shrub; this leads to a variety of plant species colonizing formerly open areas after bursage has taken root.


The purpose of our field trip was to survey the number of bursage plants in 81 square-foot plots, the distance from each bursage to its nearest neighbor, and the species of that nearest neighbor. To do so, we marked off the plot with flags and hand-counted/measured distances between the applicable plants. Our hypothesis was that bursage would be found to be predominantly sheltering other plants, rather than be the sole organism in its root zone, and that those plants would likely be another species.

My plot happened to be on a hillside with a lot of loose shale, so it was a little tricky to keep my footing and not pitch off into a ravine, but with the assistance of my lab partner we were able to get the survey done fairly quickly. After analyzing the results, we determined that we had 21 bursage plants in the measured plot. Of those 21 plants, 17 of them were found to be adjacent to another plant, which is an 81% rate of adjacency. Of those 17 plants,  5 were found to be another species that appeared to have germinated within the bursage canopy, which is a 24% sheltering rate. That sheltered species was exclusively creosote bush. This low sheltering rate did not support our hypothesis, yet procedural error cannot be ruled out as a factor in this result.

Above photo credits: Paul Cattelino. Phoenix Mountains preserve.

The dominance of bursage in the adjacency rate may be an indication of an inaccuracy in the count procedure and may have resulted in a conclusion error regarding our hypothesis. We counted each distinct crown of bursage leaves as a separate plant; however, we could not determine with 100% accuracy if each plant was a unique individual without removing the plants and conducting an analysis of the root zone(s). Thus the rate of bursage-to-bursage adjacency may be inaccurate. However, as it is already known that bursage has a distinct alleopathic root zone that prevents other plant growth when it is found to be sheltering creosote, the creosote adjacency rate appears to be accurate.

Further data is required to make an informed conclusion. Unfortunately for our lab group, only five of us showed for the field trip, so we could only make two groups and survey two plots. This makes the data we collected statistically insignificant. Nonetheless, it was fun. I enjoyed being out of the classroom for the afternoon and getting a taste of fieldwork.

That's all for now. Have a Great Week!

P.S. According to some web sources (indicated below), bursage can be used to relieve menstrual cramps and allergies. Creosote has applications, as well; it can be used as an anti-oxidant, antiseptic, and an anti-bacterial for use in minor cuts and scrapes.

Website sources to check out:

United States Bureau of Land Management

United States Department of Agriculture: Plants

Desert Eye Education

PVCC publication

Semester 4-Week 11- On Hold

Hello! Welcome back. Although last week I did promise to run some new experiments and continue primer testing, this week, like so many others do as the semester end approaches, I suddenly realized that we have three weeks of school left before finals. This means I have some catching up to do on course work, which I have spent all week doing. Additional experiments are therefore on hold, probably for the remainder of the semester.

The reason for the crunch is that I am taking a most excellent philosophy course on Bioethics, which requires that I write four essays worth 80% of my overall grade. Due to this being a late start class, the course schedule is compressed, and due dates for the papers are coming sometimes only a few days apart. So I have been knee-deep in position papers on the ethics of passive vs. active euthanasia, patient informed consent requirements, and animal research. It's a bit heady, and I am enjoying it.

There was one exciting development this week, and I must give my public thanks to S-STEM Director Amanda Chapman for making it happen. She has been in continual meetings with the ASU West faculty facilitating the transfer of S-STEM scholars into their NCUIRE program, which allows non-university undergrads the opportunity to either do authentic S-STEM experiences at ASU or begin internships at ASU as the student prepares to transfer to university. Last week, a group of PC faculty and S-STEM students toured the ASU West research labs and received an introduction to the program from Dr. Todd Sandrin, Associate Dean. It was an amazing experience.

As a result of Prof. Chapman's efforts, I met with Dr. Becky Ball, assistant professor of Sustainability Studies at the ASU West campus. Dr. Ball is a biogeochemist who studies soil conditions in multiple ecosystems (I have included a link here to her bio page). Long story short, I was offered and have accepted an internship with Dr. Ball, (contingent upon grant approval-fingers crossed!). Very exciting stuff!

The internship is not part of the NCUIRE program, however it will still provide me with the hands-on training and mentoring that I will need to continue my education and career.

Thank you, Amanda Chapman. For S-STEM, for the support you have given me over the course of this program, and for connecting me with the next stage of my development. I am in your debt.

Until next week, here is something that I hope you all enjoy:

Semester 4-Week 10- Primers and ASU West

Hello! Welcome back.

I spent lab time this week running gels on my primers to see if my initial dilutions had degraded and therefore affected my PCR results. The gels showed clear presence of nucleotide primers in my initial dilution of dry primers with TE buffer; however, results were negative for the new dilution I had made with sterile molecular-grade biology water. This result is a good indication of where I need to proceed from here, so next week will see me making new primer dilutions and doing another PCR.

The remainder of my time this week was spent attending a presentation at ASU West. We were introduced to the NCUIRE program, which allows undergrads to gain lab internship experience while receiving one-on-one mentoring (and a stipend, I might add). We were also graciously treated to a tour of the research labs, and we had the opportunity to meet with faculty researchers and question them about the parameters of their research projects.

It was an incredibly exciting experience for this uber-nerd. I am looking forward to pursuing an opportunity with the program, as this is my last semester with PC S-STEM.

Until next week, then. Happy Science! And please enjoy this brief article about mitochondrial DNA analysis that was conducted using a tissue extract from a ritually-sacrificed Incan child, circa 14,300 years ago. Click the link here.

Photo credit: Gόmez-Carballa et al./ Scientific Reports 2015. Incan child mummy.

Semester 4-Week 9-A Step Back

Hello! Thanks for joining me again.

This week, I needed to collect data on PCR amplification of the gram-negative species used for my study. Amplicons were detected post-PCR in multiple previous experiments; however, over the summer an anomaly arose after a PCR when two of the targeted species failed to amplify. This anomaly caused me concern, so I decided to re-test to see if the negative result could be repeated.

The original DNA samples I used for that PCR have degraded, so I had to perform new extractions. This is not optimal, of course. I would have preferred to re-test all of the original components, as it would be the only certain way to control the experiment, but too much time had passed since the original extraction. Additionally, I did not have enough of the original primer dilution to re-test all nine species.

From inception, the re-test was not a repeat of the original conditions that resulted in the anomaly. So while with new ingredients I could attempt to narrow down the reason for the amplification failure, there was too much variability from the summer procedure to be certain as to the cause.

This was borne out by the new PCR I ran. For the first time testing the set of PCAT-4 primers I designed for this study, amplification was unsuccessful on five of the nine tested species. I am stymied as to the reason why; there are simply too many variable changes to immediately ascertain a cause. I will have to repeat the experiment, changing one variable at a time, until I isolate the reason for the results.

That was my week. I have not had negative results for some time, and I was a little surprised (possibly even vexed) by the results. But as a wise wag (Josh James) once told me, "In science, there are no wrong answers, only new directions to explore." So I will simply explore those new directions and go where the science takes me until I find the answers I am looking for.

Cheers! See you in the lab.

PS: I just ran across a study that purports that humans emit a personalized microbial cloud that can be used to identify the unique individual. Researchers measured microbial emissions from a series of individuals and then used 16s gene sequencing to identify bacterial contributions from those subjects. It's worth a read, so I have included a link here to the published study. Enjoy!

PPS: One of the species I am targeting for amplification, Serratia marcescens:

Semester 4-Week 8

Hello! Thank you for joining me.

This week, I have been busy putting together various elements of my research paper. I had to start working on this paper at the beginning of the semester, as I needed to identify any potentially weak or missing data so that I could have time to repeat certain experiments for data clarification.

I also spent some time this week working with a fellow S-STEM scholar, Mitra. I was asked to share my extraction and gel protocols with her and give some guidance as she implemented them into her own project. We did a couple of bacterial extractions and then ran gels; unfortunately, the experiment did not yield DNA. I am not sure why this occurred. It is a procedure that I have done repeatedly in the past, so my conclusion is that there must have been an unaccounted for error in the process, and we must simply re-do the experiment. More on that on another blog.

In the meantime, I am including a link here to an article published in Yale News about the prevalence of bacteria in the indoor environment. I am also including a link here to the original paper by J. Qian, et. al, that was published in 2012. I hope you enjoy them both-they are quite interesting!

Have a Great Week!

Here's another picture of my favorite bacteria, E. coli. 

Photo credit: Centers for Disease Control, www.cdc.gov 

Semester 4-Week 7: Re-Testing for New Data

Hello! Welcome back!

This week, I have been repeating experiments I have previously done several times in order to either eliminate or account for an anomaly in my last round of gram-negative PCR amplifications. In initial testing, the primers I designed had amplified all 15 of the species chosen for my study. Multiple rounds of testing had positive post-PCR presence of DNA in my gels, but in a later phase of testing two of the gram-negatives did not amplify during PCR.

I am not certain at all why this occurred; there are a myriad of reasons which could account for the anomaly, including human error. So I did a new round of DNA extractions this week, ran some gels, and verified if DNA was present. All of the tested species were positive for DNA.

I subsequently banked the samples until next week, when I will be able to get some time on the thermocycler. Results of the PCR to follow on next week's blog.

Thanks for checking in with me.

Photo: Gel samples of 9 gram negative bacterial species showing DNA presence.

 

Semester 4-Week 6-This Semester's Research Proposal

Hello! Welcome back. Since we have research proposals due this week, I am including mine here as my blog post. Please note that as my project is wrapping up, this submission does not include the normal hypothesis/projected results format that a proposal normally would. Instead, this is a preliminary version of my project abstract. Thanks for checking in this week-I appreciate your patronage!

Abstract

Since the complete genetic sequencing of the Escherichia coli genome in 1997 (Blattner, et.al), variations of the 16s ribosomal gene have been found conserved among different bacterial species. As a result, 16s ribosomal DNA sequencing has become an integral part of bacterial identification in the modern laboratory (Janda & Abbot, 2007). The initial step of the identification process requires extraction of DNA, which must then be amplified using a polymerase chain reaction (PCR) so it can be sequenced. Primers specific to certain regions of the 16s gene are utilized to replicate the bacterial DNA during the PCR process so that the resultant DNA can be sequenced and correctly identified (Mao et. al 2012). Because PCR amplification is in widespread laboratory use, knowledge of DNA extraction and PCR techniques is an essential component of the student learner’s skill set. This study identified and designed simple laboratory protocols for use in introducing the Phoenix College student learner to elementary DNA identification technology. The protocols are intended to accompany the standard clinical format of culturing both known and unknown bacteria. The study identified electrophoresis gel techniques, extraction protocols for gram-negative and gram positive bacterial species, a set of universal 16s ribosomal primers that can be used to identify fifteen gram-negative and gram-positive bacterial species available in the Phoenix College Biosciences laboratory, and a congruent PCR amplification protocol that can be adapted and incorporated into existing instructor curricula.

References:

References: Blattner, R., Plunkett III, G., Bloch, C., Perna, N., Burland, V., Riley, M., Collado-Vides, J., & Glasner, J. (1997). The complete genome sequence of escherichia coli k-12. Science, 277, 1453-1462. doi: 10.1126/science.277.5331.1453.

Cattelino, P. (2014) Identification and application of universal 16s rRNA ribosomal primers. Phoenix College Student Paper. Pps. 1-16.

Frank, J., Reich, C., Sharma, S., Weisbaum, J., Wilson, B., & Olsen, G. (2008). Critical evaluation of two primers commonly used for amplification of bacterial 16s rRNA genes. 74(8), 2461-2470.

Janda, J., & Abbot, S. (2007). 16s rRNA gene sequencing for bacterial identification in the diagnostic laboratory: Pluses, perils, and pitfalls. Journal of Clinical Microbiology, 45(9), 2761-2764.

Mao, D., Zhou, Q., Chen, C., & Quan, Z. (2012). Coverage evaluation of universal bacterial primers using the metagenomic datasets. BMC Microbiology, 12(66), Retrieved from www.biomedcentral.com/1471-2180/12/66.

Marchesi, J., Soto, T., Weightman, A., Martin, T., Fry, J., Hiom, S., & Wade, W. (1997). Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16s rRNA. Applied and Environmental Microbiology, 64(2), 795-799.

PS: Just because I LOVE space exploration and NASA, here is a link to NASA's news page and an image of one of Jupiter's moons, Io, for your enjoyment.

This global view of Jupiter’s moon, Io, was obtained during the tenth orbit of Jupiter by NASA’s Galileo spacecraft. Credit: NASA

Semester 4-Week 5: Extraction Protocols

Hello! Welcome back.
I have had a couple of people ask me for bacterial DNA extraction protocols this semester, so I am going to publish them on this blog this week. One is an SDS thermal extraction for gram-negatives, and the second is a quick boil method for gram-positives. Feel free to copy and paste and make them your own!

Protocol#1
Spin down 500 µl of culture in a 1.5 mL tube at 12,000 rpm for 3 minutes. Repeat to increase size of base sample; total volume of sample spun down not to exceed 4.5 mL. Re-suspend cells in 0.3 mL TBS and 15 µL of 10% SDS (0.5% SDS final concentration). Incubate the solution in 55°C heat block for 10 minutes. Cool down to room temperature. Add 5 µl proteinase K solution @ dilution ratio of 5 mg powdered proteinase K per 250 µl of sterile molecular-biology grade water. Vortex ~20-30 seconds. Incubate the sample on ice for 5 minutes. Spin the sample at 12,000 rpm for 3 minutes. Verify the pellet is not loose. Transfer supernatant to a clean 1.5 mL tube. Add 300 µL of room temperature isopropanol.  Gently mix by inversion until the chromosomal DNA threads to form a visible mass. Centrifuge the DNA at 12,000 rpm for 2 minutes. Carefully pour off the supernatant, use a clean pipette to remove any remaining supernatant, and add 300 µL room temperature 70% ethanol. Invert several times to wash the DNA pellet. Centrifuge at 12,000 rpm for 2 minutes. Drain off the supernatant, use a clean pipette to remove any remaining supernatant, and allow the DNA pellet to air dry under hood for 15-20 minutes. Re-suspend the DNA pellet in 50 µL sterile molecular-biology grade water.

Protocol #2:
Incubate culture for 24 hours @ 37°C. Pellet 500 µl of culture at 12,000 x g for 3 minutes. Re -suspend pellet in 500 µl sterile water using a 30 second vortex. Incubate sample for 10 minutes @ 100°C. Pellet @ 12,000 g for 3 minutes. Pour off supernatant into 1.5 ml Eppe tube and discard pellet.
Until next week, please enjoy this National Geographic article about Homo naledi, a human ancestor recently discovered in a South African cave (artist rendition below):

National Geographic 

Photo credit: National Geographic Magazine, September 10, 2015

 

Semester 4-Week 4

Hello! Welcome back.

I am still compiling my data into a comprehensible paper, so there are no new experiments yet to report. I will only be doing lab work this semester if I run into incomplete notes on data in my lab book. If that happens, then I will need to re-run that particular experiment to complete my data set.

I was going to explain primer design on this blog, but instead I found an online resource that explains it more thoroughly than I ever could, so I have included a link to it here: http://www.cybertory.org/exercises/primerDesign/ . The lesson from Cybertory.org is one of the simplest and on-point explanations I have ever found and is a great introduction to how primers work.

I am also including a link here from Bioinformatics.org that will assist you in designing your own primers. Of course, if you would like to manually reverse-complement your DNA strands, go for it, but I prefer an online generator to reduce the chance of transposing a nucleotide and rendering my primer useless. With the Bioinformatics link, you just type in the gene sequence you are targeting, then click the function you need (reverse/complement, etc.), and your new primer sequence is immediately generated.

There are two other great resources for finding gene sequences and/or designing primers. One of them is the NBCI database, which contains an extensive listing of genomes. I have included a Youtube tutorial here (and below this post) which explains how to use the database. The home page pictured in the tutorial has changed since it was posted, but the process is still the same (the format is just slightly different). Once you find your strain of bacteria, you can simply click the "Get Primers" button and they will be provided for you, along with useful information such as the region of the gene being targeted. You can also select your parameters, as far as base pair size, by using the editing tool provided.
Another good resource is the Straininfo.net database located here. This database is particularly useful if you are using a patented strain of bacteria, are targeting the 16s rRNA gene, and are unsure of the original source strain. This was the case with my E. coli. Simply go to the link, then type your strain in the search box (for example, ATCC 4157). This will return a page that displays an overview of the strains available, and yours should be highlighted if it is in the database. After verifying that yours is available by finding the highlighted area, just navigate below the strain listing and click the "SeqRank" icon. On the bottom left of the page will be the recommended 16s rRNA sequence. Just copy and paste, edit the weird formatting so that you have a single continuous nucleotide sequence, and you are ready to analyze your gene for potential areas to target with primers.

One final word on primer design. There are other online tools available to help you design your own set that will allow you to load in genes and scan for similarites. In my case, however, I resorted to simply downloading all of the 16s rRNA sequences for the bacteria used in my study into a single Word document. Then, I looked for correlations between the genes by using the "Ctrl F" function. It was painstaking, but fun.

I hope this helps with your own primer design. See you next blog!

Semester 4-Week 3

Hello! Welcome back.

As of this blog, I am working on the initial phase of my final project paper. This phase deals with writing the "Material and Methods" section, as well as a brief summary of the outcomes of the various DNA extraction protocols I tested. I have also collected the protocols together into a series of appendices that will be included in the final result.

In between this, I have been doing an intense amount of studying. For some reason, my classes this semester require a lot of self-driven learning and repeated in-class exams. This is a bit of a change from previous semesters, so I have had to adjust my study habits accordingly.

For the science portion of this week's blog, I looked at infection rates for malaria and potential treatments. Malaria infects human erythrocytes when the organism Plasmodium falciparum is transmitted into the bloodstream via the bite of the Anopheles mosquito. While parasitic malaria infection has a wide range of symptoms, and typically presents as chills, fever, sweating, and vomiting, it is potentially fatal, particularly in those with a weakened or underdeveloped immune system. The primary method of death involves the disruption of blood flow to vital organs. According to a 2015 report by the World Health Organization, infection rates have declined by 47% globally since the year 2000, yet severe malarial cases still kill an estimated 584,000 people per year, primarily African children.

Drug-resistant strains of malaria have mutated and proven difficult to treat with conventional methods.  However, according to a study by Nobel laureate Sidney Altman recently reported on in Medical News Today by Catharine Paddock, malaria cases can be treated by disrupting the gene expression of P. falciparum by RNA. Researchers used a tool called a morpholino oligomer to disrupt expression, which resulted in slower development rates of the parasite. The lower development rates delayed the onset of full-blown infection. This new technique is a promising development in the treatment of drug-resistant strains of the infection.

I am including links to a number of articles, and the abstract for the published study. Please enjoy them!

Photo credit: National Geographic. Image: The female Anopheles mosquito is the only mosquito species that carries the malaria parasite P. falciparum.


Centers for Disease Control: http://www.cdc.gov/malaria/about/disease.html
Medical News Today article: http://www.medicalnewstoday.com/articles/299207.php
World Health Organization: http://www.who.int/mediacentre/factsheets/fs094/en/

Source for the journal publication about gene therapy:

Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum, Aprajita Garg et al., Proceedings of the National Academy of Sciences, doi:10.1073/pnas.1515864112, published online 8 September 2015

Link to the abstract: http://www.pnas.org/content/112/38/11935.abstract

Semester 4-Week 2

Hello! Welcome back.

This week was spent primarily focused on academics, but as of this writing I have also begun my final paper about this project.  This is probably going to take some time to do, as I have to sift through all of my notes from the last three semesters. I haven't always been as organized with my lab notes as I have learned to be, so I am anticipating that some of my data may be incomplete. If so, I will have to repeat portions of my project several times in order to verify the accuracy of the data that I do have.

Live and learn. Good note-keeping=the best possible accuracy.

Also this week, I spent a little time enjoying the following articles about GFP, or Green Fluorescent Protein. This particular protein was discovered in a species of jellyfish, Aequorea victoria, in the late 20th century and subsequently has proven useful for tracking protein production and gene expression in a number of animal species. The protein glows green under light in the blue-ultraviolet range, and while originally sourced from the jellyfish species, this protein was cloned in 1994 and thereafter became commercially available.

Please peruse the links. Also, please take a look at the pics; the animals shown have been altered with GFP.

http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm
http://www.uniprot.org/uniprot/P42212

Photo credits: www.buzzfeed.com

Semester Four, Week 1-Welcome Back!

Hello! Welcome back to my blog!

I am so glad to be back this semester, for my final S-STEM enrollment. I have hit the home stretch of this amazing experience, and shortly after it ends I will be heading off to ASU to complete my degree.

For the rest of this semester, though, I will be focused on compiling my project data into a coherent narrative that presents the work I have done in this program. I will still be setting up experiments, where necessary, to collect additional data, but most of my time this session you will likely find me buried in a notebook or on my laptop.

Feel free, though, to stop by and say hello. And if you are new to S-STEM, don't hesitate to contact me with any questions you have about anything. I'm here to help whenever I can.

See you in the lab!

Photo credit: FLICKR/NIAID

Here, a fractured Vero cell provides a view into a vacuole, which houses a growing cluster of Coxiella burnetii. This form of bacteria can cause Q fever, a bacterial infection that affects the heart, liver, lungs and other organs.

Summer Session-Final Post Until Fall 2015

Hello! Welcome back!

To say that it has been a busy summer would be an understatement. I had intended to take things easy, but as usual the universe had other plans for me.

I have been kept quite busy with my job this summer, so lab time was resultingly reduced. However, I think it is quite understood by the lab coordinators that, without the influx of scholarship money found during a regular semester, more work is necessary during this time to make ends meet.

I look forward to disbursement season and its renewed ability to focus on academics and lab time. I have already reduced my availability at my place of employment, and prepared my schedule for my fall tutoring gigs, in anticipation.

Until the semester begins, enjoy yourselves and this little photo of P. aeruginosa, one of my favorite subjects of my current study.

Summer Session-One Last Experiment

Hello! Welcome back!

This week, I continued repetitive testing of my P-CAT-4 primer set which had shown promise in some earlier test rounds. This was in order to verify that I am receiving the same positive PCR amplification results from separate test runs.

I performed another simple heat extraction on the gram positives (and a Pseudomonas aueruginosa strain that had been causing me some difficulty in a previous round) that I am using for this study, thawed out my previously tested gram negatives, and set up a final PCR (of the session) using that primer set. I then ran a standard electrophoresis gel @100 V for 30 minutes.

The results? Inconclusive. P. aeruginosa showed post-PCR gel banding, but there was a variation in gram-negative results from all previously performed PCRS using the samples I had backstocked. Due to these results, I ordered more stock of these gram-negative species and intend to re-test them for DNA amplification when the next semester begins as I am concerned that the samples may have been corrupted by the length of time they have been in storage.

I simply ran out of time during this session to complete this portion of the study.

Here is a P. aeruginosa gel with a ladder from this week's session; the referenced sample is all the way to the right.

Summer Session-New Primers Tested

Hello! Welcome back!

This week, I tested a set of primers which I designed last spring but had not yet used in a PCR. These primers, which I call P-CAT-3f and P-CAT-3r, were created during the same sessions that produced the P-CAT-4 primer set which I have been using to amplify DNA this summer.

As you may recall, the primer set sequences I am using were identified after I analyzed the 16s ribosomal gene maps of three gram negative bacterial strains which we have here in the lab. I then began this phase of my project by testing one primer set against nine gram negative species, including the original three used to design the primers. When several PCRs were run and DNA was amplified, I then tested the primers against six gram positive species, which also resulted in DNA amplification.

For this week's test, I first performed an extraction on the six gram positives I am using, then ran a PCR using the aforementioned P-CAT-4 set. After running a gel and verifying that DNA was present, I then took the original extraction sample and set up a new PCR using the P-CAT-3 primer set.

The results? Five of the six species amplified. The sixth, E. faecalis, did not (see gels below). Base pair sizes were approximated at 350-425 BPs long.

That's it for this week. Until next week, please enjoy this lecture from Dr. Jennifer Doudna, who co-discovered the CRISPR gene editing technique. Cheers!

https://www.youtube.com/watch?v=SuAxDVBt7kQ

Gel results:

 

Summer Session-Material Review

Hello! Welcome back to my blog. This week, I spent most of my time learning more about the technology I am using and reviewing various published papers on the nuances of the 16s gene. However, I did manage to get some time in the lab; I ran an experiment to test the effect of a lower agarose gel concentration on visual review of DNA samples under UV light. Thinner gels can increase the speed of particle travel during an electrophoresis, but what I was particularly looking for was a reduction in the "warbling effect" I have been seeing in the sample banding of my 1% gels. So I changed my concentration to 0.7 % and ran an electrophoresis to measure the results. The gel concentration did not make a difference in the banding or migration rate of the samples I tested.

From there, I moved on to running a PCR on my gram positive bacterial species using primer set 4-CAT-f and 4-CAT-r. I just wanted to repeat some earlier data by replicating previous methodology, so I cultured a new set of bacteria, performed an extraction, ran the PCR and gel-tested the results.

From the pics I took post-PCR, it appears that amplification was again successful. This is a good turn of events, as it allows me to proceed with the next phase of my project. Namely, sending my samples out to be sequenced so that I can determine which region of the gene was targeted. More on that in a later blog.

Until next week, live long, and prosper.

Photo: PCR samples; pic shows ladder in well to the left; remaining six wells correspond to gram positive bacteria used in this study.

Summer Session-PCR Results for All Targeted Species

Hello! Thanks for joining me for another installment of my blog. Your attention is very much appreciated!

This week, I continued collecting results data for my study by running another PCR on my extraction samples. This time, I decided to attempt amplification on all fifteen of the bacterial species I have been working with (a list is included below). This would be the first time I have run a PCR on all of my gram negative and gram positive samples at the same time, although I have done them in separate batches previously; I did a concurrent test this week so that I could compare the results against those obtained earlier in this project.

I also included two separate samples of P. aeruginosa that were obtained using different extraction protocols, as detailed in an earlier blog post. The purpose of this nuance in my test was to ensure that all PCR conditions were identical, so that any variance in results could be tied to the differing extraction protocols.

The results? DNA amplified; all samples appeared to be approximately 150 base pairs long. (Only two gel pictures representing eleven samples are shown due to a camera malfunction in the lab equipment.)

List of bacterial species used: S. enterica, S. sonnei, P vulgaris, P. mirabilis, P. stuartii, S. marcescens, P. aeruginosa, E. coli, K. oxytoca, B. subtilis, S. aureus, B. cereus, S. epidermidis, E. faecalis, and M. luteus. 

Summer Session-A Little Housekeeping

Hello! Welcome back.

This week, I reversed course a little and cultured some Pseudomonas aeruginosa; I then performed an extraction on this little gram negative biofilm-former using my gram positive extraction protocol, just to see if it would succeed at producing DNA. Why? You may recall that this strain was the only gram negative strain I had difficulty extracting DNA from last semester, and you may also recall that my primers were unsuccessful at amplification during last semester's PCRs as well.

After performing the boiling extraction, I ran my standard gel protocol on the sample and was pleased to find that I had visible banding present under UV. Picture below:

(Photo: Four wells, each with P. aeruginosa; wells show different concentrations of sample.)

This was good news, of course, because it allowed me to proceed with a PCR using the primers I have been testing. To date, all of the bacterial species I have PCR-tested using this particular primer set have resulted in amplified DNA post-extraction--- except for P. aeruginosa. This negative result has been gnawing at me for some time, so anything that gets me closer to wrapping up that loose end is a positive, welcome turn.

So I ran a PCR and then checked the sample using my standard gel. The photo below is what resulted.

So it appears that I got DNA. It's not pretty, but given that I blew up the cells using a boiling extraction, that is to be expected.

That's it for this week. See you on my next blog!

Summer Session-Early PCR Success

Hello! Welcome back.

Last week, I got promising results after testing two simple thermal DNA extraction methods on my gram positive species. The gels I ran post-extraction were positive for DNA banding.

Protocol #1 utilized an SDS/TBS mixture and boiling to lyse the cells and took approximately 45 minutes to set up. Protocol #2 used sterile water and boiling and took about 20 minutes from start to finish.

Bolstered by the results from both tests, I took the DNA samples obtained using protocol #2 and decided to run a quick PCR on them using a primer set that I designed last semester. As you may recall, I was able to amplify DNA from 8 out of 9 targeted gram negative species using a primer set of my own design; this experiment capped off my work last semester.

I fully did not expect to get positive results from this PCR, as my primers were designed using three gene sequence maps specific to three of the gram negatives I previously used for this study. However, after repeating the reaction using six gram positive species, DNA was successfully amplified. The gels I ran post-PCR are shown below.

This is great news! I can now move forward with the next phase of this study. Details on that to follow in a later blog.

Until then, live long and prosper.

Top: Gel 1 Bottom: Gel 2. Both gels were run using the same DNA samples. There are 6 wells loaded; from left to right the species are B. subtilis, S. aureus, B. cereus,S. epidermidis, E. faecalis, and M. luteus. Gel 1 used 2 µl of loading dye and 2 µl of sample; gel 2 used 2 µl of loading dye and 4 µl of sample.

Summer Session: First Protocols Tested

Hello! Welcome back to my blog.

This week, I began testing DNA extraction protocols for gram positive bacteria. After reading several research papers that purported in the abstract to contain a quick and simple method for extraction, I found that these postulations were not simple enough for my purposes. Therefore, I decided to create my own protocols and test them.

Protocol #1 involved pelleting down 500µl of culture (after incubating in TSB for 24 hours @ 37°C) and then re-suspending the pellet with a 30 second vortex in a 300µl TBS/15µl 10% SDS solution. I then allowed this to sit at room temperature for 20 minutes, followed by incubation in a water bath for 10 minutes @100°C.

Protocol #2 involved pelleting down 500µl of culture (after incubating in TSB for 48 hours @ 37°C) and then re-suspending the pellet with a 30 second vortex in a 500µl sterile water solution. I then incubated the sample for 10 minutes @ 100°C.

For each protocol, after incubation @ 100°C the sample was centrifuged @12,000x g for 3 minutes. The supernatant was poured off into a 1.5 ml Eppe tube and the pellet was discarded. 4µl of supernatant and 4µl of loading dye were then loaded into 10% agarose gels with 4µl SybrGreen DNA stain added to the agarose solution before setting the gels; gels were run @100 volts for 25 minutes.

Six strains of bacteria were tested for each protocol. They were: S. aureus, B. subtilis, S. epidermidis, B. cereus, Micrococcus luteus, and E. faecalis.

Visual results under UV light indicated that each protocol produced DNA, with a larger amount present in the samples taken using protocol #1. (As I am posting this blog from offsite, however, I am unable to upload the jpegs to this blog. Gel pics to follow on a later edition.)

These results are promising for a first attempt. I will be refining and re-testing these techniques when I return to the lab next week in order to duplicate my results and potentially increase my DNA yield. Until then, have an excellent weekend.

Photo: M. luteus after gram-staining. Source: Microbeworld.org

 

Summer Session-A New Course of Action

Hello! And welcome back to my blog. This post marks the beginning of my enrollment in this summer course. I was unable to blog for the past month, due to late enrollment. To catch you up on where I left off back in May:

I was in dire need of a break after the busy spring path which I had plotted for myself. After months spent tutoring three classes, mentoring the college prep class at Carl Hayden High School, interning in the lab, and working my job at the Arizona Center for Nature Conservation (The Phoenix Zoo), I decided to work full-time and loaf around while I waited for the enrollment email from Phoenix College. I got to spend a lot of time with my crazy dogs, Mr. Bentley (my pit bull) and Samuel Marshall the Third (my labradoodle). I also squeezed in a bunch of relaxation time, as well. But now it is time to get back to work. I am looking forward to it!

I have decided to move my research project into a slightly different direction. To date, I have been working solely with gram-negative bacterial species. Last semester, I tested multiple techniques for extracting DNA from gram negatives and tweaked every aspect of my project so that each procedure would maximize results from that particular type of bacteria. Even the primers which I developed for use during PCR amplification were based solely on the available 16s ribosomal gene maps of gram negative strains.

This initial focus, of course, was due to the fact that gram negative cells are easier to lyse because of their cell wall structure. Gram negatives, despite having a double-membrane, contain only a thin layer of peptidoglycan between the two membranes. This small amount of peptidoglycan can make cell lysis and DNA extraction easier; that ease of cell disruption can also lead to sheared or destroyed DNA when an extraction is conducted using a lysing additive that is too harsh for this type of cell wall. As a result, I had quite a bit of testing to do of various protocols before I settled on the simple thermal protocol using an SDS/TBS mixture which I described in last semester's research paper.

As I have at this point repeatedly extracted and amplified DNA from gram negatives, I am extending the project to include gram positives. Gram positive cells essentially contain a single cell membrane surrounded by a thick layer of peptidoglycan. This thicker layer (than that which is contained in gram negatives) can be difficult to lyse during an extraction. I have tested my gram negative extraction protocol against several gram positive strains; it was unsuccessful at producing DNA. Therefore, the first step of this summer's project will be to develop a simple extraction protocol for gram positives.

From there, I will test my gram negative nucleotide primers against the gram positive bacterial DNA to determine their viability for use with both types of bacteria. If they are proven to be unsuccessful at amplification, I will return to analysis of the 16s gene maps of all of my target species in an attempt to design new, universal PCR primers.

That is the primary focus of my summer project. I will keep you updated as to my progress.

Until next week, when I can begin posting data and photographs relevant to my project, please enjoy this pic of my dog pack. :)

Summer Session: CRISPR

This blog entry created as an interim post until course enrollment authorization is received.

Hello! Welcome back to my blog.

I recently began learning about a DNA editing technique called CRISPR. In a VERY simplified nutshell, CRISPR is a sort of immune system technique used by microbes to protect themselves against invading viruses. The microbes incorporate a portion of the invader's DNA into their own gene sequence and thereby develop a resistance to future incursions by that organism.

Some scientists successfully used the technique to make gene edits in mice and correct genetic disorders, and others are using it to improve disease resistance in crops.But what is fascinating about the CRISPR discovery is its potential for application in human medicine. Researchers have already learned how to use the technique to edit the DNA of human cells; using CRISPR, a particular portion of human DNA was removed from a cell and replaced by another sequence.

The gene-editing technique, which was developed by UC-Berkeley biochemist Jennifer Doudna and Emmanuelle Charpentier of the Helmholtz Centre for Infection Research in Germany, has enormous potential for the prevention of human disease and genetic disorders. However, the technology is so new (having been published in 2012) that the scope of its application is yet unknown.

I have attached a link to an article authored by Carl Zimmer and originally published in Quanta Magazine in February of 2015 that goes into much greater depth about CRISPR and offers a better explanation of what it can do. Please enjoy it as much as I did.

Until next week, live long, and prosper.

Article link: https://www.quantamagazine.org/20150206-crispr-dna-editor-bacteria/

Summer Session: Dermatobia hominis

This blog entry created as an interim post until course enrollment authorization is received.

Hello! Welcome back to my blog. This week's topic is about a fascinating little organism I stumbled across while doing online research into an entirely different subject, Dermatobia hominis, a.k.a. the human bot fly. Apparently, this hairy member of the Oestridae family looks rather like a bumblebee, but unlike the gentle bumble likes to incubate its larvae in living mammalian tissue. Despite its misnomer of a name, the human bot fly targets a wide range of mammals to use as a fly nursery and is not specific to human primates. That said, this fly is a familiar parasite to humans residing and travelling in its habitat, which ranges from Mexico to South America, and many a human in those areas has known the dubious pleasure of having a D. hominis larvae incubating subcutaneously for the required gestation period.

The bot fly typically lays its eggs on a mosquito, which then deliver the eggs to a mammalian host when the mosquito bites. After the egg is delivered by the mosquito, it hatches and burrows further into the host for another 6-8 weeks; after the larval stage is completed, the bot fly will self-extricate and drop off its host so that it can continue its pupal stage. This stage will often occur in any available soil.

The larval stage often appears to be nothing more than an itchy mosquito bite initially, but can easily grow to an bulbous protrusion that is approximately the size of an egg if not treated or if infection does not occur in the wound.

Simple treatments to remove the larvae usually involve asphyxiating the organisms by topical application of a smothering agent such petroleum jelly; this is allowed to sit on the wound for a day and then the larvae are removed using tweezers.

Note that I used the plural form of larva above. Yes, folks, multiple larval incubations are common in a host, particularly if the bot fly eggs were delivered via multiple mosquito bites or via another method; bot fly eggs have been known to simply drop off of trees and land in the wounds of unsuspecting hosts.

Think about that the next time you are strolling through a South American rain forest.

Until then, you can enjoy this link to a blog of a scientist who intentionally incubated a bot fly in his own skin: http://thesmallermajority.com/2015/01/09/puppy-killing-scientist-smuggles-rainforest-babies-in-body-cavity/

And you can really enjoy this video about a bot fly extraction: https://youtu.be/-K-QEEpf994

There are plenty of other fun videos showing extractions from human hosts, but I selected the one above because it is obscenity-free. Enjoy!

Photo credit: www.gizmag.com

Summer Session-Immortal HeLa Cells and Henrietta Lacks

This blog entry created as an interim post until course enrollment authorization received.

Hello! Welcome back.

I am currently reading a fascinating book called The Immortal Life of Henrietta Lacks by Rebecca Skloot. It tells the story of Henrietta Lacks, who was a poor, uneducated tobacco farmer who died in 1951 of cervical cancer. Unknown to Henrietta and without her permission, before she died her doctor took a sample of the cancerous cells from her tumor. Those cells were later determined to be immortal, or cells that could be repeatedly grown in culture without cell death; subsequently, this discovery became one of the most important research developments of the 20th century. Trillions of copies of the cells were cultured and shipped to research facilities around the world, giving birth to a multi-million dollar industry and aiding in such vital breakthroughs as a vaccine for polio. The cells have been used in every type of research imaginable, from AIDS to consumer product testing; they were the first human cells successfully cloned and were even shipped to the moon during the initial years of the space program to test the effects of zero gravity on humans.

Photo: Henrietta Lacks/Credit: http://nmaahc.si.edu/Events/BPL
Henrietta's family has never been compensated for her phenomenal contribution to science and has very little control over how her cells are utilized. The book details both her life and her family's attempts to come to terms with the legacy left by Henrietta. I'm still reading it, and I'm not writing a book report here, so I will leave further investigation into the subject to you. I have attached a couple of links that will point you in the right direction. One of them is a Wikipedia link; normally, I detest Wikipedia as a source, but in this instance the site has an extensive collection of sources that will prove useful to the reader who desires a more academic source of information about HeLa cells and Henrietta.

Good luck, and happy reading! P.S.: Please click the link below to check out a vintage video.

<iframe src="https://player.vimeo.com/video/9581140" width="500" height="375" frameborder="0" webkitallowfullscreen mozallowfullscreen allowfullscreen></iframe>

Video: Early cell progression video featuring HeLa cells. Credit: http://www.radiolab.org/story/91716-henriettas-tumor/

Photo: Multiphoton fluorescence image of HeLa cells stained with the actin binding toxin phalloidin (red), microtubules (cyan) and cell nuclei (blue). Source: directorsblog.nih.gov



Credits: All information provided by http://rebeccaskloot.com/the-immortal-life/ and http://www.livescience.com/38728-hela-cells-restricted-new-nih-plan.html .

Wiki link: https://en.wikipedia.org/wiki/HeLa

Week 14-Semester 3. Data duplicated.

Hello! Welcome back to my blog.

This week has been devoted to getting ready for the end of the semester, but I did manage to run a couple of gels on my PCR samples from last week in order to verify the results I obtained.

I am glad to report that this latest round of gels showed that DNA amplification did occur in 8 of the 9 bacterial species I tested. This is a slight change from the 9 reported last week, but still an excellent result. The reason for the discrepancy in that one organism? I suspect the original well correlating to that species that showed DNA may have simply had some bleed-over from an adjacent well. This shouldn't happen in a perfect scenario, but in a real-life lab situation, odd things and errors occasionally happen.

At any rate, this result does not yet indicate that amplification is not possible for the species in question, Pseudomonas aeruginosa. Pseudomonas differs slightly from the other organisms I am testing, in that it forms biofilms which can impede DNA extraction from the cell. In fact, during the extraction process that produced the series of samples I tested for this experiment, I did not see visible chromosomal threading during the alcohol precipitation phase of the extraction. While a lack of visible threading does not automatically indicate the absence of DNA, it is a possibility that cannot be ignored. Therefore, I must adjust the extraction protocol for the presence of biofilm, conduct another extraction, and re-run the PCR experiment on Pseudomonas a couple of more times in order to rule out the possibility that my first round of experiments may not have produced any DNA for amplification.

Such additional tests, however, will have to wait until the summer session resumes, as our last week of lab time is upon us.

Until then, have an excellent end of the semester. See you soon!

Image credit: imgbuddy.com

Week 13-Semester3. A Breakthrough!

Hello! Welcome back to my blog! Good news to report in this edition.

Last week, I had some initial success amplifying DNA from S. enterica, S. sonnei, and K. oxytoca using the first set of primers I had previously designed. As you may recall, I conducted a PCR reaction that showed positive results (for DNA amplification) and then repeated the experiment using a different set of DNA samples (in order to repeat the results and establish a pattern of amplification). Based on the data from those PCRs, this week I expanded my experiment to include the other organisms in my study. Using the same PCR protocol and primer set, I set out to see if amplification of the other species was possible.

The result? Of the nine species identified for this project, a total of five amplified during the third PCR. I considered this to be promising, as the fact that there was amplification indicated a number of things:

• The technique used to identify nucleotide sequences in the gene for potential amplification worked.
•  The annealing temperature parameters selected for the tested primers worked. 
• Given that amplification was demonstrated to be possible, testing of the other primer sets designed for this study could proceed.

This in itself was promising news. However, I still needed more data for my research paper, so I decided to expand my testing to include the next set of primers in the queue.

Therefore, I immediately set about running another PCR. I duplicated all controls from the first three reactions, exactly; even the annealing temperature remained the same, due to a similarity in optimal temperature ranges between the two sets of primers.

The results of this new test? Success! All nine of my organisms showed DNA banding in the post-PCR gels. Meaning that for the first time and after a year of testing, I was able to amplify DNA from all of my target organisms using one primer set of my own design. This, of course, is data that made me quite happy.

Also of course, this data is only preliminary; I will need to re-create the results a couple of more times, using a molecular marker, before I can be certain of the results' validity.

Looks like I know what I will be doing for the rest of the semester!

Until next week, cheers.

Paul C.

PS: Here's a link to a video about potential cloning using Mammuthus primigenius DNA.  http://www.sciencedaily.com/videos/5afc2e6cc20523ee3fc44352717bb039.htm

Photo credit: a-z-animals.com. The wooly mammoth in the collection of The Royal BC Museum, Vancouver, BC.

PPS: For more details about the work I have done this semester, including protocols, supporting data, and gel images, my research paper will be available for review via a link on this blog at the end of this semester.

Week 12-Semester 3. Finally, PCR results.

Hello! Welcome back to my blog.

This week, I was able to get lab time on the thermocycler, and thus began the final phase of my project. This last portion involves testing the nucleotide primers that I have designed for universality against the nine bacterial species selected for this project. I have already successfully tested a number of Universal Rice Primers against my extraction samples, so all that remains is to test these new primers in various combinations and at a number of different annealing temperatures and then analyze the data that provides.

Theoretically, the series of primers I am testing should successfully amplify DNA in three of my samples because the nucleotide sequences they contain were developed using the genetic sequences of those three bacteria. Therefore, I ran the first PCR of this project phase on the species S. enterica, S. sonnei, and K. oxytoca. I used an annealing temperature of 64°C during the reaction, which was squarely between my optimal primer temperatures of 62.4°C and 68.7°C. The results? DNA! However, the clarity of banding was extremely poor for S. sonnei and K. oxytoca. This may have been an indication of a number of things, including the amount of DNA present in my original extraction sample, the annealing temperature used, or the primers themselves.

In order to rule out/identify my sample itself as the potential culprit, I ran another PCR reaction using DNA samples, from a different extraction date, that were previously amplified using Universal Rice Primers. All controls in the reaction were an exact duplicate of the first PCR I ran this week.

The results? DNA, clearly visible for all three bacterial species, in my post-PCR gel.

This is a success for me on two fronts. One, it proved my hypothesis that my sample was responsible for the results of experiment #1. Two, it showed that the primers I designed work. This makes me quite happy, for it is an affirmation that my rudimentary understanding of genetics is improving.

Next week, due to these promising results, I will expand my PCR reactions to include the remaining organisms in this study. Until then, enjoy yourselves..and enjoy the following information about Neanderthal DNA that was recently extracted from an Italian specimen.

http://www.livescience.com/50458-oldest-neanderthal-dna-found.html

The above links to background information about Altamura man, a specimen of Homo neanderthalensis found in a cave in Italy approximately 150,000 years after his death.

This image shows the stalactites covering the specimen. Source: earth66.com 

Week 11-Semester 3

Hello! And welcome back.

There has been an ongoing issue these last couple of weeks with getting access to the thermocycler, so any plans to proceed with my project are on hold until that runs its course. My project has progressed to the point where all I need to do is collect data from PCRs so that I can evaluate the various primers I am using to amplify DNA; without the ability to run PCRs, all I can do is repeat what I have already done with extractions and basic lab methodology. Therefore, I have no data or conclusions to report this week.

This is actually a propitious development, as we are now nearing the last four weeks of the semester, the time when we all get slammed by course work. So although I am saddened that I cannot run reactions this week, I have instead been able to re-direct that time towards catching up assignments. This extra time is also providing a bit of relief for me because this has been my busiest semester to-date. Between the various jobs I have and the demands of academia, I have had about three days off total this entire semester. But lest one think that I am complaining, let it be known that I am grateful to be able to go to school and grateful to be in the S-STEM program.

That's it until I have some data to report. Have an Excellent Week!

P.S. Here is a picture for you, just because puppies always make people happy:

Photo credit: www.hdwallpapersinn.com

 

Week 10-Semester 3

Hello! And welcome back to my blog.

This week, I spent a great deal of time refining my extraction protocol to account for the increased growth encountered after I switched mediums from luria broth to TSB. I adjusted the initial sample size used to create a pellet for the extraction from 4.5 mL to 0.5 mL, and and reduced the amount of time that I centrifuged and vortexed each sample during the protocol application by 50% or more. This resulted in viable DNA samples for five of my eight bacterial species; the remaining three did not produce DNA. Because of the late date of the semester and the fact that I need additional data before I can write my research paper, I have decided to switch back to the growth medium I was previously using successfully for all eight species. This will allow me to get the data I need without losing any more time tinkering around with a basic procedure.

I also was able to spend some time running a PCR on one of the primer sets that I previously designed and have been excitedly waiting to test. Unfortunately, and to my profound disappointment, I have decided that the data generated from that experiment is unusable due to an error in the protocol used to provide the DNA sample used in the reaction.

I am going to re-test the reaction using another set of DNA samples that have already been evaluated for DNA using an electrophoresis gel. I have been storing the samples on ice for just such an occasion as this. The samples have been repeatedly subjected to gels, and have showed clear banding every time for all eight target species.

Sometime next week, I will set up a reaction using those samples and the primers I have designed. In the interim, please have an excellent weekend.

Please enjoy this photo of Proteus mirabilis, one of the species I am using. This little guy is commonly found in the human gastrointestinal tract, soil, and water. It can cause a number of infections in humans, and has been linked to the formation of kidney stones. (Davis & Zuber)

Photo credit:  CDC Public Health Image Library. CDC (PHIL #6691), 1976.
Link for more information from Davis & Zuber:  http://www.clinicianreviews.com/specialty-focus/nephrology/article/iproteus-mirabilisi-isolating-a-cause-of-kidney-stones.html

Week 9-Semester 3

Hello! Welcome back.

This week, I resumed the experimentation phase of my project after a brief hiatus caused by the thermocycler being unavailable. Without that essential piece of equipment, I have been unable to conduct any experiments, as the particular phase I am at within my project requires regular polymerase-chain reactions (PCRs) to test my hypothesis regarding universal primers. I did tinker around with some DNA extractions in the interim, as I am using some new base organisms for the project.

I experienced some minor hiccups in the extraction process of the new species, which after some review I have decided is a result of my switch to a new growth medium for my bacteria. To date, I had been using luria broth for cultures; with this new round of extractions, I switched to TSB. This richer medium is causing me to experience significant increases in culture growth during my standard 24-hour incubation period, which is resulting in larger pellet sizes upon centrifugation. These larger pellets were presenting some difficulty during the re-suspension step, and the enhanced vortexing required to properly mix the solution potentially resulted in an unusable extraction sample. After precipitation of the DNA was attempted using isopropanol, I had samples that were viscous and would not pellet. This caused me to not be able to retrieve a clean sample from the supernatant, and the extractions had to be discarded.

I repeated the process and adjusted the centrifuging times to account for the growth increase, which produced a less-dense sample pellet that was more easily re-suspended in solution. As a result, I was able to cleanly precipitate and isolate my DNA samples from the added bacterial species.

I then was able to proceed to the next phase of my project, which is the testing of nucleotide primers that I designed using genetic maps of the 16s ribosomal sequences of three of my target organisms. As you can imagine, I was extremely excited to finally get to this phase of the project.

I will post the results of this exciting new experiment on my very next blog, as I am still reviewing the data generated by the reaction. Until then, have a most excellent week!

Please enjoy the following abstract about the importance of 16s ribosomal gene sequencing; please download the full paper if you are so inclined:

http://www.ncbi.nlm.nih.gov/pubmed/18828852

And this:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC523561/

Plus, enjoy this representation of an enzyme used for PCRs:

Source: www.biochem.arizona.edu; DNA taq polymerase enzyme structure