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.