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Guest Blog: Do your parents understand your work?

MoBio_BlogSubmission_Graphic[1]     

My advisor always says I should ask this question to myself at every step of my research. Your work may be the best in the world. It can be innovative and exciting for you and your lab. However, you must be able to express it in simple terms, so much so that even your parents who may not come from a science background, understand what you did, why you did it, and what is next. Often, we are deeply engrossed in the complexities of our projects that we often fail to take a step back and look at it from a very simple point of view. Asking yourself whether your research makes sense to the random person allows you to critically analyse your own work and address the loose ends that might be present in your research idea, data, analyses, and finally interpretation. I also think that when striving to simplify our own words, we automatically start transitioning into trying to explain our research in a sequential and logical manner.

While I was gearing up for my defense seminar last week, I was constantly asking myself if my parents would be able to understand my research results. From the feedback I received, I did happen to do a commendable job of presenting my research which focused on “Studying Methanotrophic Bacterial Diversity in Soils, using High-Throughput Sequencing.”

I also see this theme of simplicity, underlying the EDAMAME workshop. Yes, it is an intense ten days of data analyses marathon. Yes, there are terms being said like ‘shell’ and ‘elastic cloud computing’, phrases being used like ‘Qiime-ing your time away’ and ‘honing your mothur-ing skills’, and being asked if you ‘R ready?’ But the instructors and TAs simplify it down to the basics for you. So much so that they cover super intense topics and you come out feeling like it was a walk in the park!

So go ahead and tell your parents about your work. If you are able to make them understand the ‘what’, ‘why’, and ‘how’ of your work, consider it as a job well done!

 

#simplifyscience

Written by Aditi Sengupta who recently defended her PhD in Soil Science from The Ohio State University.  Her area of research is in soil microbial ecology.

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Guest Blog: Aged Canids and Fresh Prestidigitation

There is a saying that “You can’t teach an old dog new tricks.”

I am a bit of an unusual candidate for EDAMAME2015. I am an older associate professor in the Biology Department at the University of Puget Sound, and can even remember biology prior to PCR! At the same time, I have been following the recent blossoming of research into communities of microbes over the years, ranging from hydrothermal vents, to insect guts, to acidic cave effluent, to the ever present human microbiome. Sociomicrobiology seems to be everywhere in science today, and I find the topic fascinating.

As part of my job teaching microbiology at the University of Puget Sound, I began introducing students to the concept of what kinds of microbes appeared to live in their reusable water bottles years ago—at first via simple streaking and observation, to colony PCR with universal 16S PCR primers and phylogeny, and finally a tentative foray into next generation sequencing: real complexity. This brought home central concepts in microbiology, as well as applications to everyday life.

At the same time, I began to explore microbiology-centered research collaborations with two of my colleagues at the University of Puget Sound. The first involved sex-specific differences in the cloacal microbiota of a species of lizard in Southern Arizona. The second involved fermentation-generated seeps of hydrogen sulfide rich seawater in nearby Commencement Bay, resulting in microbial communities similar to those found at deep sea hydrothermal vents. In both cases, it was necessary for me to better understand changes in the structure of microbial communities. It quickly became clear that I lacked the tools and training.

I had been struggling for a while with these studies, with occasional help from patient scientists such as Jack Gilbert at Argonne National Laboratory. Progress was slow and frustrating. So when the announcement of EDAMAME2015 appeared, I decided to apply, despite the fact that I am, um, a quite a bit older and less experienced than most folks taking the course.

It has been a wonderful experience. The lead instructor, Ashley Shade, was patient, supportive, and funny (FIGURE 1). The talented and friendly teaching assistants never once rolled their eyes at me as I learned about line commands, the shell, the eternal quandary of QIIME versus mothur, Prokka, MG-RAST, and the arcane secrets of GitHub. The instructors never gave up on me, even when I sputtered to an intellectual stop regarding R. The instructors, and my fellow students, pitched in and brought me up to speed. It was truly an educational community.

Figure01[4]

I tweeted out lots of highs from EDAMAME2015, such as my victories over mothur (FIGURE 3), and short-lived triumph over R (FIGURE 4).

Figure03[3] Figure03[4]

Figure04[1]

 

The tutorial materials and experiences have certainly clarified much of what I am doing back in Tacoma, and given me a great deal of insight into improved approaches! I think that, because of this course, I can finally feel more “in control” of my collaborative projects. Also, I am thinking of how I might use these skills in my Fall microbiology course, letting each student analyze the microbial communities within their water bottles (funding permitting, of course) (FIGURE 5)!

Figure05[1]

After a long day of tutorials and pecking at the keyboards, we were treated to quite an impressive array of seminars with expertise relevant to the skills we were trying to learn or hone. The speakers included Vince Young , Jay Lennon, Ariane Peralta, Jim Cole, and Jim Tiedje. We had the chance to chat and socialize a bit with each of the speakers; what an opportunity!

I haven’t mentioned the nonscientific aspects of EDAMAME2015. The course was located at the Kellogg Biological Station near Kalmazoo, Michigan. And as the photographs (FIGURES 6, 7, and 8) show, it is a lovely locale for learning.

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Did I mention the fireflies at night? In addition, the students were diverse, warm, helpful, and welcoming. They enjoyed my tardigrade bottle opener (FIGURE 9), and even made me microbially themed origami (FIGURE 10). One night, we went on an expedition to a lovely brew pub in Kalamazoo (FIGURE 11), while another night we enjoyed a great barbeque (FIGURE 12).

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Figure10

 

Figure11

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I believe that I have made some new friends and colleagues.

While I will be sad to see the end of EDAMAME2015 approach, I also know that I return to Tacoma armed with knowledge, skills, and a network of new colleagues who have already proved their willingness to help out people new to this area of research.

Yes, there is a saying that “you can’t teach an old dog new tricks.” To which I would quote Henry Ford: “Anyone who stops learning is old, whether 20 or 80.” EDAMAME2015 was just the place to remind this particular aged canid that I can still learn and grow as a scientist.

It is truly a transformative experience for people interested in microbial ecology, and I highly recommend it as an intensive, positive, and unforgettable experience.

 

Mark O. Martin, PhD

Associate Professor of Biology

University of Puget Sound

E-mail:       mowenmartin@gmail.com

Twitter:      @markowenmartin

Blog:          microbesrule.blogspot.com

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EDAMAME 2.0!

We received overwhelmingly positive feedback from our Explorations in Data Analysis for Metagenomic Advances in Microbial Ecology (EDAMAME) workshop last year. It was immediately obvious that we were addressing an urgent unmet need in the scientific community. The data from our course evaluation showed that EDAMAME learners had achieved our overarching goals of increased confidence and competence in microbial sequencing data analysis. In short, we seemed to be doing a lot right and having a positive impact on our learners’ abilities to own their microbial sequencing analyses.

We were ecstatic.

This year, we had double the number of applicants from last year, and many of them were so exceptional that distinguishing among them for the purposes of admissions was quite challenging. We have reached out to recruit a broader applicant pool than last year, and I see the benefits of our efforts reflected in the diversity of backgrounds and academic interests represented among our EDAMAME learners this year. We also had a portion of our applicant pool from governmental organizations like the USGS and EPA, and also from not-for-profit organizations, which reflects a need beyond academia for the flavor of training that we provide. In retrospect, the high level of interest from many different universities, institutions and agencies makes sense: microbes are the functional foundations for all ecosystems, from human bodies to soils to deep-sea vents. Why shouldn’t there be wide interest in learning how to observe and analyze Earth’s ubiquitous but functionally elusive microbial communities?

At EDAMAME, we strive to provide the best learning materials and instruction that we can, and there is always room for improvement! For #edamame2015, we’ve refined our learning objectives (listed at the bottom of this post) and backwards-designed tutorials to meet those objectives. We’ve also spent more time on topics we glossed over last year like starting, using, and transferring files to an Amazon EC2 instance, which we hope will help learners who do not have access to a high performance computing cluster to know how to access the computing resources needed to execute analysis of our ever-larger sequencing datasets. After feedback for “MORE TIME” from last year’s EDAMAME learners, we have expanded to 10 days so that we can spend more time with the more complex material. We also have scheduled time for independent study with instructors available to give learners the opportunity to analyze their own datasets with our support. We also organized our tutorials in a GitHub wiki (with a CC-BY license) so that folks outside of the course can more easily find and use our materials.

And, this year, with almost a full year behind me on the tenure-track at Michigan State University, I also was able to bring with me my own new team of students and post-docs to serve as teaching assistants for the course. They bring contagious enthusiasm, patience, and experience to the course (they took EDAMAME in its inaugural year in 2014, even before joining my team). At every break, we collect “minute cards” (borrowed form Software Carpentry best practices) to receive immediate feedback from learners on what is going well and what needs to be addressed. With this feedback, our learners have “spoken” loudly: our TAs are just amazing. I am so lucky to be supported by these stellar young scientists.

Three days in to the course, we have already covered navigating the shell, cloud computing, remote sessions for running long jobs, within-sample and comparative diversity, merging paired end MiSeq reads and QIIME for microbial amplicon analysis. Today we are using mothur for amplicon analysis, and in the next few days we will be learning shotgun metagenome analysis (assessing quality, digital normalization, assembly, annotation), R for ecological statistics, RDP tools including their new exciting targeted gene assembler Xander, and using public databases. And other useful computing stuff. J We’ve also got a great line up of guest speakers, starting with Vince Young from the University of Michigan last night. During breaks, there will be volleyball and campfires and the backdrop of the serene Kellogg Biological Station on Gull Lake. It is going to be legendary!

I am actively writing grants and seeking support of EDAMAME in future years, but right now we are uncertain as to what the future holds. If Culture Dish readers know of any funding opportunities for microbial metagenomic / computation training of graduate students, post-docs, faculty, and research scientists from around the world, please reach out and let me know at shadeash AT msu.edu.

Signing off from summery Michigan State University’s Kellogg Biological Station,

EDAMAME onward!

Ashley Shade @ashley17061

 

EDAMAME 2015 Learning Goals

  1. Increase computing literacy
  2. Develop proficiency in cloud computing
  3. Analyze microbial amplicon sequences
  4. Analyze microbial shotgun metagenome sequences
  5. Apply ecological statistics to analyze and interpret microbial sequencing data
  6. Access resources provided by public sequence databases
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EDAMAME 2015

It’s a bird, it’s a plane, nope!  It’s time once again for EDAMAME (Explorations in Data Analysis for Metagenomic Advances in Microbial Ecology)!

 

edamame

We are so stoked to host another series of blogs in the upcoming weeks!  Please stay tuned for the ultimate science reading experience.

Go get ‘em EDAMAME!

 

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BiOstic® Bacteremia…Our Dirty Little Secret

Apr 10, 2015
Michelle Tetreault Carlson

bacteriaInBlood

When the BiOstic® Bacteremia DNA Isolation kit was originally designed, it was optimized for the extraction of bacterial DNA from infected blood. However, it has turned out to be useful for so much more. Bacteremia is the presence of bacteria in the blood and under normal circumstances blood should be sterile, but with the insertion of a catheter into the body or through an open wound, bacteria can gain entry and take hold.

Even a small numbers of bacteria can be a real problem and make people very sick. Hospitals need to have a low bacterial detection threshold, and in order to achieve this the blood is first cultured, which can take 48 hours and is applicable only to culturable microbes. To complicate things further, some patients have already been dosed with antibiotics or other drugs which can interfere with the culturing process.

The basic premise of the BiOstic® Bacteremia DNA Isolation Kit is to isolate DNA from a small number of bacteria inside a messy mixture of blood and anticoagulants or growth media. Standard blood or microbial kits won’t work for this. Microbial cells are harder to lyse than blood cells and the heme and lipids in the blood can be potent PCR inhibitors which need to be removed using specialized chemistry.

The BiOstic® Bacteremia DNA Isolation kit combines bead beating and detergent for complete bacterial cell lysis with MO BIO’s patented Inhibitor Removal Technology®, for the removal of heme molecules and other inhibitors from the DNA. The first steps in the BiOstic® Bacteremia DNA protocol are to remove a portion of cultured blood; centrifuge to pellet all of the cells and then bead beat the cell pellet with a strong detergent-based lysis buffer. The kit uses 2 ml MicroBead tubes containing fine garnet sand and is ideal for lysing microbial cells. Bead beating is followed by MO BIO’s Inhibitor Removal Technology®.This is the same powerful chemistry that is used in our soil and fecal kits and will remove all PCR inhibitors including heme, lipids, polysaccharides, humic acid, and heparin.

New Applications

Isolating microbial DNA from cultured blood is a pretty specific application. Soon after the BiOstic® Bacteremia DNA Kit was released, we found other situations where it was useful to get DNA from a dirty cell pellet. Customers started trying it on all kinds of other samples and it turned out to work fabulously. We thought it time to let you in on our dirty little secret.

Dirty Swabs

Swab samples which contain PCR inhibitors (fecal, vaginal, wound, intestinal/stomach, environmental surfaces) tend to contain a relatively small number of cells in a hot mess of PCR inhibitors. The BiOstic® Bacteremia Kit’s strong lysis buffer combined with its one step Inhibitor Removal Technologyâ turns out to be ideal for maximizing the DNA yields. To use a swab with this kit, start with the following protocol:

  1. Add 450 µl of the CB1 buffer directly to the MicroBead tube.
  2. Place and rotate the swab in the buffer and let it soak for a few minutes to release the cells into the solution.
  3. If the swab has a head that can be snapped off, go ahead and do so, leaving the swab in the tube. Otherwise remove the swab now while gently squeezing it against the wall of the tube to remove as much of the solution as possible.
  4. Proceed with the 70 C heating step and the rest of the normal protocol.

Dirty Microbial Pellets

Any situation where bacteria can be pelleted from a dirty (containing PCR inhibiters) liquid can work well with the BiOstic® Bacteremia DNA Isolation kit. Some examples include sputum, saliva, washes from dirty surfaces (nasopharyngeal and colorectal), and mixed culture isolates. To utilize these types of samples with this kit, follow this protocol:

  1. Centrifuge the liquid at 13,000 xg for 2 minutes to pellet the cells. The volume of liquid will depend on the sample. Aim for a wet cell pellet weight of 25 mg or less.
  2. Remove the supernatant.
  3. Resuspend the cell pellet in 450 µl of the CB1 buffer and add to the MicroBead tube.
  4. Proceed with the 70 C heating step and the rest of the normal protocol.

DIRECT FROM (uncultured) BLOOD

Customers often want to avoid culturing blood because of the time and possible bias involved. The kit can also be used for the isolation of bacterial DNA directly from blood without culturing, but less starting sample has to be used in order to avoid clogging of the spin column. To use the kit for DNA isolation from whole blood, follow this protocol:

  1. Centrifuge 50-500 µl of whole blood at 13,000 xg for w minutes to pellet all of the cells.
  2. Pipette off the supernatant and dispose of as hazardous waste.
  3. Resuspend the cell pellet in 450 µl of the CB1 buffer and add to the MicroBead tube.
  4. Proceed with the 70 C heating step and the rest of the normal protocol.

And More…

In addition to the sample types mentioned above, the BiOstic® Bacteremia Kit has been used for filtered plant washes, feces and even raw milk. We’ve listed some recent references below. Maybe you’ll come up with more ideas.

So what started as a bacteremia kit is now much more – should we ask the Marketing Team to rename this product: PowerDirtyLittleSecret? Let us know you opinion!

References

Direct from Blood:

Identification of Different Bartonella Species in the Cattle Tail Louse (Haematopinus quadripertusus) and in Cattle Blood
Ricardo Gutiérrez, Liron Cohen, Danny Morick, Kosta Y. Mumcuoglu, Shimon Harrus, and Yuval Gottlieb
Appl. Envir. Microbiol., Sep 2014; 80: 5477 – 5483.

From Cultured Infected Tissue:

Mupirocin and Chlorhexidine Resistance in Staphylococcus aureus in Patients with Community-Onset Skin and Soft Tissue Infections
Stephanie A. Fritz, Patrick G. Hogan, Bernard C. Camins, Ali J. Ainsworth, Carol Patrick, Madeline S. Martin, Melissa J. Krauss, Marcela Rodriguez, and Carey-Ann D. Burnham
Antimicrob. Agents Chemother., Jan 2013; 57: 559 – 568.

A Serologic Correlate of Protective Immunity Against Community-OnsetStaphylococcus aureus Infection
Stephanie A. Fritz, Kristin M. Tiemann, Patrick G. Hogan, Emma K. Epplin, Marcela Rodriguez, Duha N. Al-Zubeidi, Juliane Bubeck Wardenburg, and David A. Hunstad
Clinical Infectious Diseases, Jun 2013; 56: 1554 – 1561.

Direct from Swabs:

Rectal Swabs Are Suitable for Quantifying the Carriage Load of KPC-Producing Carbapenem-Resistant Enterobacteriaceae
A. Lerner, J. Romano, I. Chmelnitsky, S. Navon-Venezia, R. Edgar, and Y. Carmeli
Antimicrob. Agents Chemother., Mar 2013; 57: 1474 – 1479.

Extravaginal Reservoirs of Vaginal Bacteria as Risk Factors for Incident Bacterial Vaginosis
Jeanne M. Marrazzo, Tina L. Fiedler, Sujatha Srinivasan, Katherine K. Thomas, Congzhou Liu, Daisy Ko, Hu Xie, Misty Saracino, and David N. Fredricks
The Journal of Infectious Disease, May 2012; 205: 1580 – 1588.

Filtered plant washes:

Ecological Succession and Stochastic Variation in the Assembly ofArabidopsis thaliana Phyllosphere Communities
Loïs Maignien, Emelia A. DeForce, Meghan E. Chafee, A. Murat Eren, and Sheri L. Simmons
mBio, Jan 2014; 5: e00682-13.\

Direct from Saliva:

Salivary Microbiota and Metabolome Associated with Celiac Disease
Ruggiero Francavilla, Danilo Ercolini, Maria Piccolo, Lucia Vannini, Sonya Siragusa, Francesca De Filippis, Ilaria De Pasquale, Raffaella Di Cagno, Michele Di Toma, Giorgia Gozzi, Diana I. Serrazanetti, Maria De Angelis, and Marco Gobbetti
Appl. Envir. Microbiol., Jun 2014; 80: 3416 – 3425.

Direct from feces:

Membership and Behavior of Ultra-Low-Diversity Pathogen Communities Present in the Gut of Humans during Prolonged Critical Illness
Alexander Zaborin, Daniel Smith, Kevin Garfield, John Quensen, Baddr Shakhsheer, Matthew Kade, Matthew Tirrell, James Tiedje, Jack A. Gilbert, Olga Zaborina, and John C. Alverdy
mBio, Sep 2014; 5: e01361-14.

Direct from Raw Milk:

“Remake” by High-Throughput Sequencing of the Microbiota Involved in the Production of Water Buffalo Mozzarella Cheese
Danilo Ercolini, Francesca De Filippis, Antonietta La Storia, and Michele Iacono
Appl. Envir. Microbiol., Nov 2012; 78: 8142 – 8145.

Blood Culture:

Herbaspirillum Species Bacteremia in a Pediatric Oncology Patient
Edward D. Ziga, Todd Druley, and Carey-Ann D. Burnham
J. Clin. Microbiol., Nov 2010; 48: 4320 – 4321.

 

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Become BFFs With Your FFPE

Apr 02, 2015
Michelle Tetreault Carlson

Formalin-Fixed, Paraffin-Embedded (FFPE) tissues, the most common tissue preparation method for archiving bio-specimens, can be a valuable resource for genetic studies. The fixation process makes it possible for samples to be stored for years at room temperature, for analysis even decades later.

Chemical fixatives like formalin (a mixture of formaldehyde and methanol) preserve the structural integrity and morphology of the tissue by cross-linking neighboring amino groups between proteins. This traps other molecules like carbohydrates, lipids and nucleic acids in place.

While the fixation process preserves the structural integrity of the cells, it also denatures proteins and causes the degradation of RNA and DNA. The longer the fixation process and the age of the tissue the more damaged the nucleic acids.  As a result, the size of the nucleic acid fragments is generally small, in the 100-500 bp range. All this makes the extraction, amplification and analysis of nucleic acids a bit tricky. However, using appropriate protocols and kits it’s still possible and highly worth the effort.

MO BIO has two awesome kits for isolating DNA or RNA from formalin fixed paraffin embedded (FFPE) tissue. The traditional method for removing wax from FFPE tissue has always been to use xylene, a highly flammable and toxic organic solvent. The tissue is washed several times in xylene to dissolve the wax and then the xylene is removed by performing multiple washes with ethanol before doing the DNA isolation. This results in lots of extra handling where the tissue is repeatedly washed. Each time a wash occurs; some of the sample can be lost along with the wax and the solvent.

The BiOstic® FFPE DNA and RNA kits avoid such harsh treatments by leaving the wax intact for the extraction. By using an optimal combination of denaturing buffers and higher temperatures, the activity of proteinase K can be optimized so that complete digestion of the tissue occurs while the wax is melting. This reduces handling time and loss of tissue.

In this example (provided by a customer) using 10 micron thick single slices removed from histology slides, the samples in blue were pre-processed with xylene and DNA isolated following the manufacturer’s protocol and the BiOstic FFPE DNA samples were extracted. Yields were quantified on the Nanodrop.

In this example (provided by a customer) using 10 micron thick single slices removed from histology slides, the samples in blue were pre-processed with xylene and DNA isolated following the manufacturer’s protocol and the BiOstic FFPE DNA samples were extracted. Yields were quantified on the Nanodrop.

Both kits follow similar protocols.1 to 5 slices of FFPE tissue (or up to 15 mg) are digested with Proteinase K at low heat in order to liquefy the tissue and release the DNA.  Then a second heating step at higher temperature is used to remove cross-links that can inhibit PCR or other enigmatic reactions.

Because RNA degradation can be more severe for these sample types, the BiOstic® FFPE RNA Isolation Kit uses a gentler, pH neutral lysis buffer and the Proteinase K digest and the cross-link removal are done at lower temperatures and for shorter times.  We also added our new low elution silica spin filter with a final elution volume of only 20 µl, to this kit in order to increase the final RNA concentration.

Comparative analysis of the BiOstic® FFPE Tissue RNA Isolation Kit (MB) and competitors Q and LT of RNA extraction from a single 10 micron tissue slice of FFPE Normal Human Liver tissue. a)  1.2% TAE gel showing higher yields of intact RNA obtained when sample was prepared with the BiOstic® FFPE Tissue RNA Isolation Kit. b) Invitrogen Qubit™ Fluorometer readings show that higher yields of RNA were obtained when sample was prepared with the BiOstic® FFPE Tissue RNA Isolation Kit (MB).

Comparative analysis of the BiOstic® FFPE Tissue RNA Isolation Kit (MB) and competitors Q and LT of RNA extraction from a single 10 micron tissue slice of FFPE Normal Human Liver tissue. a) 1.2% TAE gel showing higher yields of intact RNA obtained when sample was prepared with the BiOstic® FFPE Tissue RNA Isolation Kit. b) Invitrogen Qubit™ Fluorometer readings show that higher yields of RNA were obtained when sample was prepared with the BiOstic® FFPE Tissue RNA Isolation Kit (MB).

FFPE tissues are a unique sample type with a lot of challenges, but when it comes to DNA or RNA isolation, we’ve made that part easy.

Samples are available and can be ordered on the web or upon request at no charge if you call/email customer service. If you have more questions such as how to remove C/T artifacts that could occur at a very low rate in some tissue, technical support is here to help. We’re waiting to help you out!

Some recent references for the BiOstic® FFPE DNA Isolation kit:

TERT promoter mutations are associated with distant metastases in papillary thyroid carcinoma
Greta Gandolfi, Moira Ragazzi, Andrea Frasoldati, Simonetta Piana, Alessia Ciarrocchi, and Valentina Sancisi
Eur. J. Endocrinol., Feb 2015; 172: 403 – 413.

Microbial communities present in the lower respiratory tract of clinically healthy birds in Pakistan
Muhammad Zubair Shabbir, Tyler Malys, Yury V. Ivanov, Jihye Park, Muhammad Abu Bakr Shabbir, Masood Rabbani, Tahir Yaqub, and Eric Thomas Harvill
Poultry Science, Feb 2015; 10.3382/ps/pev010.

Anaplastic Lymphoma Kinase–Positive Large B-Cell Lymphoma: Description of a Case With an Unexpected Clinical Outcome
Magda Zanelli, Riccardo Valli, Isabella Capodanno, Moira Ragazzi, and Stefano Ascani
International Journal of Surgical Pathology, Feb 2015; 23: 78 – 83.

 

 

 

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Whatcha Been Up To?

We are starting a new series of blogs that will include a short synopsis of a recently published peer-reviewed paper. We want to keep you (and ourselves!) up to date on the latest and greatest science news.

Psst, don’t forget about our Published Reference program, you get a free kit for sending us (reference@mobio.com) your published paper using our kits!  Not a bad gig!

mobio-blog-emelia

This month we picked:

Metagenomic Analysis of the Airborne Environment in Urban Spaces

Nicholas A. Be & James B. Thissen & Viacheslav Y. Fofanov & Jonathan E. Allen & Mark Rojas & George Golovko & Yuriy Fofanov & Heather Koshinsky & Crystal J. Jaing

This group collected samples by filtering air that trapped airborne microorganisms from urban spaces in Washington, DC. They extracted the nucleic acids off the filters and then used next-gen sequencing to characterize the community. This work is increasingly relevant in recent times of bio-terrorism. It is therefore very important to be able track this kind of activity and their paper describes a method to do so.

biohazard2

What’s even more interesting is that their experimental design included areas that had been purposely colonized by spore forming Bacillus thuringiensis serovar kurstaki, a known pesticide against gypsy moths. B. thuringiensis is non-pathogenic to humans but because it is so closely related to Bacillus anthracis, the agent that causes anthrax, it can act as a “pathogen surrogate” to determine the detection of bio-terrorism.

The bottom line is that they found a seasonal pattern (spring, summer, fall, winter) in the data and there was sufficient detection of the spore-forming organism. Therefore, they created a protocol that can be used for detection of potential aerosolized bio-terrorism activities.

Thanks for using MO BIO and please keep up the awesome science work, we love to learn and be inspired!!

 

 

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The Origin of Life on Earth and Aliens

Mar 02, 2015
Emelia

Hello MO BIO! IMG_3866

My name is Jesse McNichol and I’m a graduate student in the MIT/Woods Hole Joint Program and along with my supervisor, Dr. Stefan Sievert, I study the microbiology of deep-sea hydrothermal vents. These ecosystems are extremely unusual on Earth, since they are mostly supported by volcanic activity instead of the sun’s light. The oxidation of hydrogen sulfide and hydrogen gas are probably the ultimate source of food for all the teeming life around these hot springs, including the large fishes, tube worms and crabs.20141105135543

 

I’d seen these ethereal ecosystems before through a video monitor, and I was fortunate enough to see them up close and personal in during our research expedition in November 2014 from the window of the submarine Alvin! Seeing the huge black smoker chimneys and the giant tube worms reminded me why I became so fascinated with vents in the first place. Because they are so different and mostly independent of sunlight, many people (myself included) study them to better understand how life might exist on other planets, such as in the oceans of extraterrestrial moons Europa or Enceladus.

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Although a fascination with life on other planets is what brought me into this field, the motivations for this study are more ‘down to earth’. While the genomic revolution has given us an incredible amount of detail on which organisms exist in deep-sea vents, we actually know very little information about them. How fast do they grow? How much energy they need to survive? How do they interact with one another? In short, we have a very incomplete picture of the ecology of these unique deep-sea environments.

 

Being at the bottom of the ocean, under over 250 atmospheres of pressure, these ecosystems are naturally very difficult to sample. That’s why I’m fortunate to work at Woods Hole – not only do we have access to the R/V Atlantis and the submarine Alvin, my supervisor’s long-time collaborator Dr. Jeff Seewald has designed and pioneered a sampling device that can bring to the surface an accurate ‘snapshot’ of the deep-sea community, and keep the microbes under the intense pressure found in the deep sea. Although it had been used routinely before for chemical sampling, I was the lucky graduate student to get to try growing microbes for the first time in Dr. Seewald’s samplers.

IMG_6634

Given this incredible opportunity, I spent a lot of time thinking about what conditions to test to unlock the unsolved mysteries of these ecosystems. Eventually, I carried out 53 high-pressure incubations during two oceanographic cruises in 2014, which continue to yield new insights as I process the chemical and genetic data. In the end, this work will yield clear estimates on the productivity of the microbial community at deep-sea hydrothermal vents, which will help us understand the ecology of the whole system.

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For the general public, the scientific details will not be as important, but I hope that when people see how much more we have to learn about these ecosystems on Earth, it will inspire them to learn more about what I think is the greatest story yet to be uncovered – how life arose and evolved on Earth and how it could exist outside of our small planet.

IMG_1616

To support the telling of these stories, MO BIO plays a key role. Although I often still use ‘old school’ DNA extraction techniques, DNA and RNA extraction kits are essential for our everyday work in the lab. We use them to generate high-quality DNA and RNA for experiments that help us to understand these organisms and their ecology better, especially the effect of different chemical conditions on the activity and expression of poorly understood metabolic genes.

IMG_1620To read more about the November 2014 Research expedition click here for the official website and here for an article written about Jesse McNichol.

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Inspiring Women Scientists in Microbiology and Ecology: Part Deux

Feb 12, 2015
Emelia

There’s been some social media buzz lately about #WomenInScience. No better time than the present to update the list from our previous blog!

 

Inspired by #ISME15

Recipient of the ISME Young Investigators Award:

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Ruth E. Ley is an Assistant Professor of Microbiology at Cornell University in Ithaca, NY. She was trained in ecology and natural history at the University of California Berkeley (B.A.) and in ecosystem science and soil microbial ecology at the University of Colorado, Boulder, where she worked with Dr. Steve Schmidt (Ph.D.). Her post doctoral research was first with Dr. Norman Pace, working on highly diverse hypersaline microbial mats. She then transitioned to working with Dr. Jeffrey Gordon on the microbial ecology of obesity at Washington University School of Medicine. She is an author on 4 of the 10 most highly cited papers on the human microbiome. Her interdisciplinary group at Cornell works on the human microbiome at different scales of analysis, including large-scale genetic studies in human to discover novel pathways of interaction between host and microbiome, and mechanistic studies of those interactions using germfree mice as a tool to assemble and interrogate specific microbiotas. Dr. Ley’s awards have included the Hartwell Investigator Award, the Arnold and Mabel Beckman Young Investigator Award, a David and Lucile Packard Foundation Fellowship, and an NIH Director’s New Innovator Award.

Keynote Speaker at ISME:

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Julia Vorholt is Professor of Microbiology at the Swiss Federal Institute of Technology Zurich (ETH Zurich), Switzerland. She carried out her Ph.D. work at the Max Planck Institute for Terrestrial Microbiology in Marburg, Germany. After a postdoctoral stay at the University of Washington, Seattle, USA, she returned to the MPI in Marburg as a group leader and subsequently headed a group at the Centre National de la Recherche Scientifique in Toulouse, France. Since 2006 Julia Vorholt is Professor at the Institute of Microbiology of ETH Zurich. She investigates how the environment, in particular the phyllosphere, shapes bacterial physiology, with an emphasis on metabolism, novel protein function and gene regulation. She applies metaproteogenomics to bacterial phyllosphere communities, uses synthetic bacterial communities to study microbe-microbe-plant interactions and develops Fluidic Force Microscopy for single cell analyses. She received the Otto-Hahn medal of the Max-Planck Society and is a member of the German National Academy of Sciences, Leopoldina.

Inspired by #MOBIO Customers:

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Emma Allen-Vercoe

In her own words:

I began my research career with undergraduate and graduate studies at the Central Veterinary Laboratories (now Veterinary Laboratories Agency) and the Centre for Applied and Microbiological Research (CAMR, now the Health Protection Agency), UK, under the direction of Prof. Martin Woodward. There, I studied the enteric pathogen Salmonella enterica serovar Enteritidis, and developed a sound appreciation of the many obstacles that a enteric pathogen must overcome in the gut in order to cause disease. I became fascinated by the huge arsenal of virulence factors required by enteric pathogens in order to survive and proliferate in the gut environment.

I spent a brief postdoctoral period at CAMR, learning to work with technically challenging pathogens such as Mycobacterium tuberculosis and Campylobacter jejuni, before I relocated to Canada in 2001 to start a postdoctoral position at the University of Calgary, under the joint direction of Drs. Rebekah DeVinney and Mike Surette. Here I worked on Enteropathogenic and Enterohemorrhagic E. coli (EPEC and EHEC), using cell and molecular biology techniques to probe the fascinating interactions of their type III secretion systems with host cells.

I had always been interested in learning more about the normal microbial population inside the human gut, and in 2004 I was fortunate enough to win a Fellow-to-Faculty Transition award through the Canadian Association of Gastroenterology. This award allowed me to develop an independent research program aimed at the study of the normal human microbiota and its influence on human health and disease, a program that I brought with me to Guelph in December 2007.

My motto: “My microbes told me to do it”—->  (We at MO BIO are all going to start using that as an excuse all day everyday!)

My hobbies: Gardening, reading, reading about gardening

Thanks Ladies for all the inspiration!  We are honored at MO BIO to serve you…

Want to see more rockin #WomenInScience, #SciWomen, #WomenInSTEM, #GeniusWomen?

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Protect your RNA samples during DNase treatment

The DNase step is one of the most common causes of degradation or loss of the RNA during your extraction. DNase digestion is frequently performed on the spin column and although this can be a great way to save  time on the post extraction processing, it is not an efficient method for samples with large amounts of DNA (for example, spleen, thymus, and even some soils). In these cases, DNase digestion in solution is necessary.

The typical protocols for DNase involve inactivation of the enzyme using EDTA and heat. Both of these things can cause problems in RT-PCR. EDTA can inhibit the RT-PCR enzymes and heating the RNA can cause a reduction in integrity.  Additionally, most DNase enzymes are stored frozen and need to be aliquoted to avoid freeze/thaw cycles that can reduce enzyme efficiency.

We came up with a better system that protects the RNA all the way to the final step: DNase Max.

DNase Max is a liquid room temperature stable DNase with very high activity (1 ul of enzyme can digest 30 ug of DNA in 20 minutes). The best part is the clean up step. The DNase comes with a highly specific removal resin that binds the enzyme and cations and pulls them out of the RNA sample making it ready to use in qRT-PCR without any inhibitory additives or heat steps.  The resin is so efficient that completely removes 10 units of enzyme in the reaction, (figure below, lanes 3-4) compared to an alternative resin method incapable of removing the just 2 units of DNase enzyme used (lanes 1-2).

This means that you can protect your precious RNA as well as hours of work invested and get better accuracy in gene expression assays.

DNase MaxRemoval Resin completely removes DNase. Samples were subjected to DNase treatment and enzyme removal using the  DNase Max™ Kit or a competitor’s kit, and then analyzed for residual DNase activity using the MO BIO DNase-free certification assay. Lane 5 is the negative control and did not receive DNase. Samples were incubated for 1 hour at 37C, followed by inactivation for 5 minutes at 65C. Results are shown on a 1% agarose gel. The DNase Max Removal Resin successfully removed the DNase (lanes 3-4), while the competitor’s resin failed to remove all of the DNase from the samples (lanes 1-2).

Other tools to protect your RNA.

Isolation of RNA, no matter what the source, is nerve wracking, but especially when samples are limited or irreplaceable.  Because RNA is so labile, working quickly but carefully is the key. There are ways to protect your RNA during the procedure so that you can work at a relaxed pace and without so much anxiety.

The use of BME, consistent and fast homogenization, and RNase-free DNase with removal resin will be your ticket to success in every prep no matter what the sample. Certified RNase-free gloves are a great extra to have as well as UltraClean Lab Cleaner for removal of nucleases from the bench and equipment. We use these routinely in our labs.

 

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