Why is molecular research on microbes in water difficult?

For some people, getting back to the original source of water may not be possible for months or even years. For example, we talk to scientists collecting samples at hydrothermal vents in the middle of the ocean, in the Antarctic, and in the Baltic Sea.  For some researchers, water samples may have been collected after a certain event, such as a flood or heavy rain and so the conditions of the water will not be the same in a week or even after a day. They need to get answers from every sample collected and they need it to accurately reflect the current microbial content.

Choosing a Filter:

People who want to determine the microbial communities of collected water will filter them onto filter membranes. The typical size is a 47 mm membrane. This is large enough to have a good flow but small enough to work for DNA or RNA extraction. If the membrane is too small (25 mm), it may clog if the water contains higher levels of debris and if it is too big (142 mm), it will need to be sliced up in order to fit in standard 5 ml and 15 ml tubes.  Ideally, the less handling and manipulations going on with the water filter, the more microbial DNA and RNA can be recovered.

To help make sure that the 47 mm filter membranes are extracted the most efficiently without needing to be sliced into small pieces, MO BIO Labs uses a 5 ml screw cap tube (see picture right). This tube allows for full access of the microbial side of the filter to be homogenized with the garnet grinding resin. We have found after thorough testing that this tube allows for maximal recovery of DNA from all types of filter membranes.

Another question we hear from customers is how to choose a type of membrane. There are many choices from polyethersulfone (PES) to mixed cellulose esther, MCE (cellulose acetate and cellulose nitrate) to polycarbonate to aluminum oxide. Each of these membrane types handle a bit differently and will give slightly different results after extraction.  It is important to remember that the different characteristics of a membrane also reflect its use for other applications such as direct culturing (PES, MCE) or light and electron microscopy (polycarbonate, aluminum oxide).  Overall selection of a membrane for DNA and RNA isolation is more dependent on pore size, sample volume, and retention of inhibitors such as pesticides.  In other words, more than one membrane type may work for your application.

In our experience here is what we found:

Polyethersulfone: Are one of the toughest membranes and can be handled more than the others. They dry quickly under vacuum making them easy to fold without tearing.   Both 0.45 and 0.22 micron pore sizes can be used but a 0.22 micron pore size is best when you want to filter large volumes of water with low microbial biomass because they can handle the longer harder pressure of the vacuum. For nucleic acid extraction, we can get yields equivalent to the mixed cellulose esther with the PowerWater® DNA and RNA Isolation Kits.

Mixed cellulose esther (cellulose acetate and cellulose nitrate): Are best for when a 0.45 micron pore size is needed.  We recommend the use 0.45 micron pore size if your water has a lot of debris and tends to clog or filter very slowly with 0.22 micron pore sized membrane. Cellulose membranes tend to retain water making them a little more difficult to handle.  The video below will demonstrate how we handle them in our lab.

There are several published studies demonstrating that pesticides and herbicides can bind to cellulose acetate and cellulose nitrate so if you are using water that may contain pesticides and herbicides, avoid using cellulose membranes.

Polycarbonate: This type of filter can be more difficult to work with due to its thinness and the ease at which it can wrinkle.  A 0.45 micron pore size is commonly used to prevent clogging.  Unlike the PES and MCE membranes, microbes in your water sample will sit on top of the membrane rather then inside.  This leads to clogging faster but also retention of smaller particles that would have been able to pass through.  We have found that for isolating DNA, less extreme bead beating will give you higher molecular weight DNA. If your sample is used for PCR only, then the stronger bead methods should be fine although expect a lot of shearing.

Aluminum Oxide: This type of filter is also known as an Anodisc™ filter membrane (Whatman).   It handles like a thin sheet of glass and will break up easily in any bead tube. Most labs are not using these due to the difficulty in transferring them to storage tubes. These are used with samples containing very low biomass such as ocean water.  They come in both 0.45 and 0.22 micron sizes.  Similar to the polycarbonate, microbes are retained more on top rather than within the filter, leading to easy extraction of DNA and RNA but also increased shearing with bead beating.

How to Handle a Filter Membrane:

Many of you out there probably already have a good technique for folding your filter membranes and placing them into a tube. For those of you who are new, or experiencing problems with this, here is a video we made in our lab to demonstrate the technique. This is a 0.45 micron mixed cellulose esther membrane.  Demonstrating is Heather Callahan, Ph.D, the scientist who created the PowerWater^® kits.

Summary:

We know how hard it is to work with different environmental water samples and how important it is to get every last drop of DNA or RNA. Our goal is to make sure you are successful at every step. When we developed the PowerWater and RapidWater products, we kept that in mind as we optimized each step; from the tube needed to grind in, to the matrix used for grinding, to the solutions used for removing inhibitors, to the binding chemistry, to the washing chemistry, and the final elution. If you have any questions on how to maximize your water filter DNA or RNA extraction, please send them to us and we’ll get back to you.  I can assure you, if you have a problem, we have the answer.

Ask the Water DNA and RNA Isolation Experts!

For more information on Water DNA and RNA Isolation products, go to:

PowerWater^® DNA Isolation Kits and RapidWater^® DNA Isolation Kits

PowerWater® RNA Isolation Kits

*This article is a re-post of the original which was published in October of 2009

Fun fact: Shakespeare's sonnets have been encoded in DNA

I would like to meet whoever is responsible for perpetuating the myth that scientists are a stuffy and dry bunch and introduce him or her to the amazingly creative and artistic scientists we have the pleasure of interacting with at MO BIO.  This month we invited our users to submit a poem about their MO BIO experience. The results were quite impressive. The topics ranged from our kit’s unparalleled ability to remove inhibitors to our great customer service. In return for their very kind words, we offered up a t-shirt and discount code.

Be sure to check out the video links of MO BIO employee’s reading the submitted poems.

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We start off our MO BIO Poetry Corner with a poem from our CFO/owner, Liz Brolaski. Mark Brolaski, CEO/owner, reads Liz’s poem that gives a shout out to the awesome employees at MO BIO.

A poem by Liz Brolaski
MO BIO Laboratories, Inc.

MO BIO rocks
And let me tell you why
The people who work here
Are pleasant as pie

They come to work each day
With a smile on their faces
Even when they’re dreaming
of far off places

So if you think it’s just the products (and services)
That make this place great
Think again my friend, you’ll see
It’s the crew, they are TOP RATE!

***************

This poem should be sung to the tune of ‘Soft Kitty’ from the show, The Big Bang Theory.

Check out Michelle (Technical Support Manager) and Mary Jo (Customer Service Manager) singing their hearts out:

A poem by Katerina Fagan-Solis
North Carolina Central University

Fast Service
Awesome Products
MO BIO Laboratories
Quicker, cleaner isolations
No more agonies!

***************

This poem is sure to resonate with anyone that has struggled to isolate DNA from water samples run through Sterivex filter units. Of course, this is before finding MO BIO’s PowerWater Sterivex DNA Isolation Kit. Heather C. (R&D) and Amber (Lab Services) dramatically orate Carmelita’s poem:

Sterivex Isolation
A poem by Carmelita Jaramillo

Filter after filter.
Fifty mls of flow.
Sometimes worried that the filter might flow too slow.
High DNA yield is double stranded gold.
At the right salt concentration the column won’t let go.
No cause for alarm.
Heating to sixty five won’t cause any harm.
Use the solution while it’s warm.
Transform.
Whole cell to cell contents.
Lysis.
Combination of mechanical and chemical disruption.
Homogenization.
Centrifugation.
Reduce impurities that inhibit downstream DNA applications.

***************

Our next poem expresses a customer’s great happiness for clean DNA.

Katie’s (Distributor Customer Service) moving rendition of the poem can be found here:

A poem by Anonymous

I feel so happy when using this kit today,
I know I can depend on getting lots of clean DNA
I love MO BIO more than words can say…

It comes in a box, so pretty and blue!
I can’t resist this discount, it’s true!

***************

We believe that this is the first time humic acids have been mentioned in any piece of creative writing–excellent work, Zackary!

Christina (Customer Service) does an impressive job of bringing this poem to life:

A poem by Zackary James
Colorado School of Mines

MO BIO is the place to be
DNA extractions are hassle free
Inhibition is a thing of the past
Keep those humic acids and give me those nucleotides

***************

This poem is an ode to the much beloved PowerSoil DNA Isolation Kit. Do you have a difficult sample type, too? We offer free samples of our most popular kits here.

Lily’s (Lab Services) riveting reading is absolutely a must view:

A MO BIO Poem
A poem by Lauren Henry
Northeastern University

DNA to extract but I was so weary
Of a lack of bands on my agarose gel
No kit to break me from my own private hell

My algae cells of thecae thick
Would yield to nothing, it made me sick
No chemicals or great technique
Could save me from this life so bleak

Yet from the heavens there came a cry,
Or perhaps maybe it came from my PI
She said unto me, “do not despair!
Give one more try, I’m sure we’ll get there!”

A MO BIO kit, the PowerSoil, promised results
And so I did toil.
And to my delight, when the extraction was done,
My PCR finished, and the gel run

I saw glowing bands of bright DNA!!
MO BIO, you’ve done it! You have saved the day!

***************

Our last rhyme honors our TB Dry Powder Growth Media. Our very own Erika (Customer Service) does her best reading of it here.

A poem by Liz S.

My dear to whom it may concern,
I write today with hopes to earn
a discount from your company
in exchange for my dignity

A calendar hanging by my station
ignored by the rest of our vocation
was picked up by our boss who then
declared to me “You write to them”

I must admit, your TB’s great
for increasing final product weight
and when my cells are growing fine
it takes a huge weight off my mind

So like a heart-struck kid I’ll say
from your TB I’ll never stray,
and that nothing else works quite as well so I don’t even mind the smell

The others here could share their views
and thoughts on products that they use
but they’d rather see me stumble through an attempt to write a poem for you

So bottom-lined, I love your stuff
and simply can’t express enough
how much your TB has helped improve
our product and thus too our mood

***************

Thank you to everyone that submitted a creative piece this month. Have your own poem to share? We would love to hear it, leave it in the comments or e-mail us at customercare@mobio.com for your own shirt and promo code!

Debates on this subject have kept philosophers busy for centuries, lending to rich discussions on everything from evolution of chickens to the beginning of life and the universe. One could ponder this question from a literal point of view and discuss the evolution of egg laying species and whether or not this pre-dates the appearance of chickens. Or one could set forth on a metaphysical journey and focus on the possibilities of how one life form can exist without its developmental precursor or how a precursor exist without its original maker?

Here at MO BIO, this discussion led us down a different path. The question we propose to you is: Which came first, the DNA or the protein? You need DNA to know what proteins to make and how to make them, but, you need proteins to synthesize more DNA, to transcribe the DNA into RNA, and to hold it all together.

So which came first? The instruction manual or the technician? The recipes or the cook? Love or marriage? Oh wait, scratch that last one…

Today’s post is to encourage you to think about this question. We focus so much on DNA and RNA and what this tells us about diversity and function, but what about the proteins? There is an interplay between the two and we can’t have one without the other. But…. which came first?

Vote here and enter to win one of MO BIO’s very cool new t-shirts!

And if you need help coming up with an answer, some very interesting philosophical discussions are here, here, and here.

Here’s the scene: You have a precious soil sample collected from the roots of an ancient never-before seen orchid located on a remote island in the middle of the South Pacific. You isolated the DNA from microbes in this soil using a method other than the PowerSoil Kit. It’s still dirty and won’t amplify in PCR so it needs to be cleaned of humic acids and other humic substances. You need every last molecule for whole genome shotgun sequencing…. what do you do?

You call MO BIO and explain your situation and we recommend the PowerClean Kit. The PowerClean method is a mini-version of the PowerSoil Kit without bead tubes. You run the sample through the same process using IRT and isolate clean DNA. But wait… the spec readings have changed! Before clean up it said you have 300 ng/ul of DNA and now it says you have 30 ng/ul.  It’s your worst nightmare. Your DNA has been lost!

Hold up. Slow down. We’re here to tell you, your DNA is safe and sound- and CLEAN. There’s some confusion about how to tell what you had before and after clean up using standard quantification lab techniques. We’re glad you called us.

Let us explain….

I want to go back to a previous article we wrote about substances that are co-extracted with DNA from environmental samples that wreak havoc on UV260 wavelength readings and cause false yield information.  This is key because many of these organic inhibitors from soil, water, biofilms, and plants co-absorb at the 260 wavelength and interfere with accuracy in the DNA yield readings (as well as PCR amplification). So does degraded RNA which will be isolated if using CTAB or phenol:chloroform methods. And so do silica spin kits that do not have a method for inhibitor removal and use very strong binding salts that do not discriminate between DNA and RNA binding. All of these factors are going to cause problems with yield data interpretation.

The good news is, once these interferences are removed, the new yield readings are the TRUE yield reading.

Let’s see a real life example of how this will look using standard laboratory techniques; the Nanodrop and an agarose gel electrophoresis.  Beginning with the Nanodrop, below we see the results of the same soil extracted with and without inhibitor removal technology (IRT). The first thing everyone looks at when QCing their DNA prep is the ng/ul yield reading. This is important but this is only a small part of the story.

Take a look across the cells…

The 260/230 ratios tell us that there is a problem with this DNA. In the samples with inhibitors, the readings are below 1.0, indicating a high level of impurities. We can get another key piece of information from this data- the 340 reading. Look at how elevated they are compared to the samples below them. They are almost 10X higher. Humic acids will optimally absorb at wavelength 320 so the high absorbance at 340 reflects the carry over of humic acids in the final DNA. The 260/230 ratio, combined with the high 230 absorbance, tells us this DNA is not clean and it is caused by organic compounds.

Nanodrop also provides graphical representation of the absorbance data across all of the wavelengths.  This too is very handy. We clearly see a high level of interference across all of the wavelengths being measured. It starts high and stays amplified. The 260 reading is caught in middle of this inhibitor-fest and way above where it should be.

However, we know that this is still difficult for some people to believe, that this is not all DNA. So the best way to confirm your results is an agarose gel picture.  The gel picture has information you cannot see on the Nanodrop; integrity of the DNA and a visual representation of the yields. Now we see a different story. Although the Nanodrop says the Non-IRT samples are more than double the yields of the IRT samples, the gel picture shows us they are actually less.  The contaminants make analysis confusing. This is why we always recommend checking your DNA with two methods – Nanodrop along with a gel picture or picogreen reading.

So what happens when you clean dirty DNA samples up using the PowerClean Kit? You remove these interfering compounds from the DNA and as a result, you see a drop in the yield reading. A big drop.  In this case it would be more than 50%. But it is NOT the loss of high molecular weight DNA. It is loss of the compounds you don’t want in your prep; pesky PCR inhibitors costing you time and money!  It is now pure DNA.

Let’s take a deeper look at this second contributor to falsely elevated DNA yields: RNA

Depending on the method used, RNA will co-extract with the DNA. It may not be intact, but if it is there it will absorb UV.  Any method using phenol or chloroform to extract nucleic acids will co-isolate the RNA and methods using strong binding salts and ethanol in equal volume will co-isolate RNA. Because the Nanodrop cannot differentiate between DNA and RNA, this is where a second method such as Picogreen comes in handy, which measures DNA only. An agarose gel picture is also helpful because the RNA smear can be visualized.

In this article, The Difference Between the Nanodrop and Fluorescent Dye for Quantification of DNA, we show how much RNA can impact your yield readings.

In this example using a plasmid prep, when using 4 ml of overnight LB E.coli culture, we can very easily see the degraded RNA smear at the bottom of the agarose gel run samples above (lanes 4-6). A comparison of the Nanodrop vs. Picogreen readings are shown on the right.  Based on the Picogreen data, DNA yields are more than 60% lower compared to the Nanodrop reading.  RNA absorbance can impact readings as much 70% for some samples.  This amount of inaccuracy in your DNA sample can lead to pretty big errors later on.

The good news is that MO BIO’s DNA Kits are designed to bind DNA and not RNA, ensuring an accurate reading on the Nanodrop, as you saw in the figure above.  And binding conditions are easily adjusted to increase or decrease the sizes of nucleic acid captured if small fragments or RNA is desired.

So the take home message is this: Look at your DNA sample before clean up and then after clean up on an agarose gel. Take a look at the intensity (yield) and integrity (size range of the DNA) and compare with your second method (Nanodrop or Picogreen) to check for the presence of RNA or inhibitors.

You can save yourself anxiety and time if you do a simple check to see what you are starting with. Remember these kits can only give back what you put in.  You want clean DNA.  But knowing how much DNA you had to begin with will calm your nerves and give you the assurance you need about the outcome.

We’ve all got our groove on. Sampling and processing seems like a breeze despite it’s challenges.  The flow of efficiency has finally set in and it feels good!

Last week we dropped off the birding duo at Avian Island, a “Antarctic Specially Protected Area” where you need special permits to land.   With the help of a few shipmates, they set up a camp and spent five days researching the massive Adelie penguin colonies breeding there.  During the set-up (which took about a half day), others were able to take the small boat to a deserted Chilean Base for some exploration.  It is a relief to be on solid ground and take an afternoon off, boosts morale especially when you have the opportunity to listen to the sounds of the elephant seals that we encountered.

Think growling, whoopee pad, and burps happening simultaneously and amplified by a hundred.  Honestly, I can’t help but tell you it is hysterical, mesmerizing, and remarkable all at the same time.  I will never again feel embarrassed about the decibel of my own bodily vibrations!

A few days later, I finally got the tap on the shoulder, one I had been anticipating for some time.  The whalers invited me to help sample humpbacks using a crossbow, I was super stoked!   Don the Mustang Survival gear and go hunting for whales.  Not your everyday activity.  After several hours, we had 4 samples and were covered in whale snot which, as you can imagine, smells rancid from the strictly seafood diet maintained by the ginormous creatures.

Our next stop?  Rothera Station owned and run by the British Antarctic Survey.  We were welcomed like brothers and sisters and treated to a day of genuine merriment albeit the rivalrous annual soccer game.  We lost 0-1.  Crevassing, afternoon tea, and a decadent dinner rounded out the day.  Perfect.

Now the next charge was to get to our furthest point South, Charcot Island.  This island is named after the father of the famous French Antarctic expeditionist Jean-Baptiste Charcot.  This proved a tricky maneuver for the Laurence M Gould and the crew navigating her.  The area is more sea ice than not, and with any change in the wind direction, the ice can shift leaving you pinned in.  This happened in 2001 close to Palmer Station.  The team was stuck for a month to wait for the ice to subside.  Our second attempt was a success and the birders could complete their data set by counting the Adelie colony present on the island.  It was a celebratory day, successful landing, hitting the furthest Southern point, and the icing on the cake?  It was my birthday.

From here on, we will travel North as we near the end of our tenancy in these waters.

In a week, we will be back at Palmer Station off loading shipmates (including myself) and taking on new folks for the passage through the Drake and ultimately back home.  I will take the Holland America’s MS Prinsendam cruise liner to Buenos Aires because of space constraints (bad life eh?) and will leave a few days after the Gould.  In as much, I am told that I will partake in the Palmer tradition of “polar plunging” as she leaves the dock to go North.  I am already shaking in my steel-toed XTRA TUF boots quite literally.

This concludes my literary stories from The Last Frontier.  Thanks for the tremendous amount of support, I have been honored to share my journey with you.  In the words of Dr. Suess, “Don’t cry because it’s over, smile because it happened!”

~Cheers,

Emelia

Photo Captions in order as they appear:

Adelie Penguin (Cameron Rutt):  Adelie Penguin

Elephant Seals (Zach Swaim):  The loud and vocal elephant seals.

Crevassing at Rothera (Natasja van Gestel):  Returning to British based Rothera Station after crevassing on the glacier.

Avian Island camp (Cameron Rutt):  The Birder’s living quarters on Avian Island.

Below: Avian Island penguins (Cameron Rutt):  The massive numbers of Adelie penguins on Avian Island

Since many of our customers are now turning their efforts towards extracting RNA from more difficult samples (AKA dirtier), we figured it would be a good time to talk about some of our newer RNA kits:  PowerMicrobiome RNA, PowerPlant RNA, PowerWater RNA and PowerBiofilm RNA.  All four of these RNA Isolation kits use the same combination of two solutions to bind the RNA in your cell lysate to a silica spin column.  It’s the ratio of these two binding solutions that determines the size of the RNA that will bind and subsequently the size of the RNA you extract.  Everything smaller than the cutoff point will wash off the column.  Everything above the cutoff will stay on the column and will come out in your elution.   You can alter the binding solutions to change the cutoff point but you’ll always lose everything smaller than the cutoff.

So how can this be used to your advantage?   First, let’s talk about the sizes and types of RNA that are in cells.   The ribosomal RNA (rRNA) will be by far the most abundant type of RNA you isolate.  That’s because rRNA makes up about 80% of the total RNA in each cell.  In prokaryotic cells you’ll find three major varieties of rRNA:  23S (2906 nt), 16S (1542 nt) & 5S (120 nt).  In eukaryotic cells you’ll find 28S (5070 nt), 18S (1869 nt), 5.8S (156nt) and 5S (121 nt).  If you run your sample on a standard 1% agarose gel, you can see the two largest rRNA bands running side-by-side.  The second most abundant (~15%) RNA in cells is transfer RNA (tRNA).   The tRNA is used to translate codons to amino acids in the ribosome.   It’s pretty small, ranging from between 70 and 90 nucleotides.

The third most common RNA, and probably the target for most of our customers, is messenger RNA (mRNA).  The mRNA is used to convey the genetic code for proteins from the genome to the ribosomes.   Therefore its size is as varied as the proteins it encodes for.  Although most of it will be in the 2000 base range, you can find mRNA as small as 500 and as large as 14,000 bases.  Unfortunately, this variation makes it difficult to select for it by size.   On a gel, it looks like a faint smear mixed in with the rRNA.  Virtually all of the rest of the RNA in the cell consists of microRNA (miRNA, ~22 nucleotides), used for gene regulation in eukaryotic cells or small interfering RNA (siRNA, 20-25 bp), which impedes the expression of some genes.

Okay, so here’s the scoop.  If you follow the standard kit protocol, you’ll isolate all RNA greater than around 60 nucleotides.   This means you will get rRNA, mRNA and tRNA but lose the miRNA and siRNA. To pull in smaller RNA you’ll need to lower the size cut-off by altering the binding mixture.   The standard binding mixture is 1 volume of binding solution (PM3, PR3, PWR3, or BFR4, depending on the kit), 1 vol of 100% ethanol and 1 volume of lysate, a 1:1:1 mixture. To lower the size cutoff, you’ll need to add an additional volume of 100% ethanol, so a 1:2:1 mixture.  This will allow you to isolate pretty much all of the RNA that’s there.  If you want to raise the size cutoff, you’ll need to replace the solution containing 100% ethanol (PM4, PR4, PWR4, or BFR5, depending on the kit) with 70% ethanol.    This will give you just mRNA and the two largest rRNAs.   If you are studying mRNA, this helps reduce the background RNA a little bit.

We thought this might be a litte confusing.  So we’ve created a handy chart to help.   You can use the alternate bind with any of the kits in the chart.  Each of these kits also includes our strongest inhibitor removal technology (IRT).   Note that you cannot use this technique with our RNA PowerSoil kit or the UltraClean Microbial RNA kits.  If you have any questions regarding the technique please contact us at technical support:  technical@mobio.com

References:  Molecular Cell Biology. 4th edition.  Lodish H, Berk A, Zipursky SL, et al.  New York: W. H. Freeman; 2000.

You may have heard some buzz floating around recently about the guy who cleverly proposed to his girlfriend via agarose gel. Yes, we know, it’s incredible.  We wanted to ask him a few more questions about this amazing, geeky proposal,  so we tracked down the proposer, Eric (currently a Post-Doc at UCSD) , to get the whole story.

How did you two meet?

We were both graduate students at Stanford. We met at a grad student event and played volleyball together. We ran into each other again later at a department happy hour.

Where did you get the idea from?

I wanted to come up with something fun related to what we both do. Romantic-y stuff didn’t really fit us—a little bit geeky fit us better. I guess the idea came at a campus football game, I was looking at the scoreboard and thought about the idea of writing with a limited set of lines. I had the idea for a few months and couldn’t think of a better one, so I stuck with that.

Did everyone in your lab know?

Actually only one other person in my lab knew, and she also helped me with picking primer pairs.  I wanted to pick a time when the lab would be empty, so it could be more personal.

How did you actually design the gel?

I initially did a mock-up in Illustrator on the computer first (just copying and pasting bands to design it), and used that to figure out what sizes of DNA I needed. Once I settled on 5 sized PCR fragments (roughly 150, 300, 500, 700, 1kb), I went back through my notes to find 5 primer pairs that I knew worked pretty well from previous experiments. The other lanes are just mixes of the 5 sizes (either 2:3:4 or 4:6 volume mixes going in decreasing size, since larger fragments tend to be brighter). The gel actually didn’t take that long (though it was terrifying loading it), the hardest part was keeping all the mixtures organized so I could follow my pre-designed sketch without mis-loading something (which wouldn’t have been fun!).

How did you actually get her to image the gel?

There was a lab holiday party that night, so I told her I had forgotten my gift and that I had to go back home. I had run the gel far enough and had checked it to make sure it looked good and then called her to ask her to image it for me. She was actually in such a science mind-set that she called me to tell me that the gel looked funny—no ladder and a lot of fragments. When I asked her to go back and take one more picture, she happened to turn the image sideways and realized what it was.

Was the proposal a surprise? Did she say yes?

No, we had actually picked out a ring earlier. And yes, she did!

What are your respective areas of focus?

I am working on RNA binding proteins and their roles in neuronal stem cells and neurodegenerative diseases and she’s in cancer biology (studying a p53 target gene).

******

We’d like to thank Eric and Jeanine again for answering all of our questions about this agarose gel proposal! We wish them both the best!

So, we want to know, do you have any adorable and science-y proposal stories to share? Happy Valentine’s Day to everyone (even if you’ll just be sitting in lab watching your E. coli grow).

As I was pondering potential speakers, I thougth about the women in science I’ve heard speak, who are an inspiration to me. But my experience is also limited being in the private sector. I don’t get to interact with as many women thought leaders as I would like. The question then became: who are the leading women in the field right now?  What women scientists do we have to draw insight and inspiration from?

I attend the Gordon Research Conference in Applied Microbiology every other year where I get to meet so many outstanding scientists from across the globe and of course ISME (also every other year) is a chance to network with many in Europe who don’t make it to ASM each year. ASM is a great meeting as well, however, we know many environmental science labs save their money for smaller more intimate meetings and avoid the crowds of ASM.

So I turned to my community on twitter to help me come up with a list of inspirational women scientists in environmental microbiology and ecology that have made an impact in their careers.  Who are the top women in the field right now? Who is it that we would love to see speak?

Here is what we have so far. What would I love to see happen next? Wouldn’t it be great if we could get all of these women together in one forum where women in science could interact, ask questions, and hear how they navigated the path to success?   Does anyone think this could happen at ASM in Denver this year?

And if you are a graduate student in microbiology and ecology and looking for a mentor for your next step, perhaps one of these PIs has a place for you.  In alphabetical order by last name, here are the women we think are inspirational women in science!

Ginger Armbrust University of Washington
Research focus: Phytoplankton, how organic carbon generated by phytoplankton is used by bacteria and archaea, and their interactions with other microbes.

Jill Banfield University of California, Berekely
Research focus: Geomicrobiology, how microorganisms shape and are shaped by their natural environments, microbial dissolution and precipitation of minerals, the structure, properties, and reactivity of nanoparticles (many of which are formed by microorganisms), microbial ecology, and microbial evolution.

Bonnie Bassler Princeton University
Research focus: Quorum sensing and development of anti-microbial drugs aimed at bacteria that use quorum sensing to control virulence, and improved industrial production of natural products such as antibiotics

Liane Benning University of Leeds
Research focus: Quantitative elucidation of biogeochemical reactions at low to hydrothermal temperatures and in both, inorganic and biologic systems. The mechanisms and kinetics of mineral nucleation and growth and the associated speciation, sequestration or release/transport of various elements in Earth surface environments; the preservation and adaptations of microbial life in extreme environments in geothermal and arctic settings.

Antje Boetius Max Planck Institute for Marine Microbiology
Research focus: Microbial ecology of the deep sea, marine methane cycle, gas hydrates and cold seeps, geomicrobiology, and global carbon cycle.

Colleen Cavanaugh Harvard University
Research focus: Symbioses of bacteria in marine invertebrates from deep-sea hydrothermal vents, methane seeps, and coastal reducing sediments. Specific emphasis on characterization of metabolic and genetic capabilities of symbionts, evolutionary relationships with free-living bacteria, and co-evolution of host and symbiont.

Penny Chisholm MIT
Research focus: Ecology, evolution, and comparative genomics of marine cyanobacteria and the viruses that infect them; Iron and phytoplankton growth; Ocean fertility.

Rita Colwell Chairman of Canon U.S. Life Sciences, Distinguished Professor at University of Maryland, College Park
Research focus: Global infectious diseases, water, and health, developing an international network to address emerging infectious diseases and water issues, including safe drinking water for both the developed and developing world.

Pascale Cossert Pasteur Institute
Research focus: Molecular and cellular basis of the infection by Listeria monocytogenes, a model pathogen for the study of the intracellular parasitism.

Angela Douglas Cornell University
Research focus: Interaction between animal function and the diversity and activities of resident microorganisms.

Nicole Dubillier Max Planck Institute for Marine Microbiology
Research focus: Symbionts in marine invertebrates, transfer of energy from the geosphere to the biosphere at hydrothermal vents and cold seeps, and ecology of chemosynthetic environments.

Katrina Edwards University of Southern California
Research focus: Geomicrobiology and microbial life at or below the sea floor, and the interaction of microbes and rock. Director of the National Science Foundation supported Center for Dark Energy Biosphere Investigations.

Mary Firestone University of California, Berkeley
Research focus: Interactions of bacteria with the soil environment, microbial community ecology of N and C cycling, mechanisms of interaction between plant roots and soil microorganisms.

Susan Gottesman National Cancer Institute, Laboratory of Molecular Biology
Research focus: Small Regulatory RNAs and Energy-Dependent Proteolysis: Novel Modes for the Regulation of Gene Expression

Jessica Green University of Oregon, Twitter: @JessicaLeeGreen
Research focus: Built environment microbiome, host-microbe systems biology, microbial metagenomics

Carol Gross University of California, San Francisco
Research focus: Regulatory mechanisms of E. coli stress responses, protein interactions in the bacterial transcription apparatus, and genome-wide control of gene expression.

Jo Handelsman Yale School of Medicine
Research focus: Diversity in microbial communities and the role of microbial communities in infectious disease. In particular, understanding a gut community that serves as a source of opportunistic pathogens, and the soil microbial community as a source of new genes and proteins.

Christine Jacobs-Wagner Yale University
Research focus:  Temporal and spatial mechanisms involved in bacterial physiology, with emphasis on chromosome dynamics, cell division, cell cycle regulation, cell morphogenesis and RNA biology. Our primary model organisms are Caulobacter crescentus and Escherichia coli.

Janet Jansson Lawrence Berkeley National Labs
Research focus: Molecular microbial ecology and “omics” approaches with a focus on soil, marine sediment and the human gut environments.

Hilary Lappin-Scott Swansea University, Wales, Twitter: @lappinscott
Research focus: Unravelling the complexities within microbial biofilm communities, co-founder of ISME Journal, President of the Society for General Microbiology.

Margaret McFall-Ngai University of Wisconsin, Madison
Research focus:   Host responses to interactions with beneficial microbes including how environmentally rare bacteria are harvested from the host’s habitat during the onset of a horizontally transmitted symbiosis, by what mechanisms does the host recognize its specific symbiotic partner,  what influences symbiotic bacteria on the developmental of the host tissues with which they associate, how symbiont population is maintained over the host’s lifetime, such that neither the symbiont overgrows the host nor does the host eliminate the symbiont, and what  similarities and differences exist between pathogenic and beneficial animal-bacterial interactions.

Trina McMahon University of Wisconsin, Madison
Research focus: Microbial ecology, environmental biotechnology, biochemical engineering, environmental engineering, water quality, metabolic engineering  and freshwater microbiology.

Mary Ann Moran University of Georgia
Research focus: Sulfer cycle gene discovery, environmental transcriptomics, understanding specific metabolic functions of marine bacterioplankton taxon and their importance in assimilating and mineralizing carbon and nutrients in seawater, Roseobacter model organism system development.

Nancy Moran Yale University
Research focus: Symbiosis, particularly between multicellular hosts and microbes

Dianne Newman California Institute of Technology
Research focus: Molecular mechanisms that underlie microbial formation and/or dissolution of minerals, understand the chemical signatures of early life found in the geologic record, and quantitate/control the contribution of microbes to present-day geochemical cycles.

Victoria Orphan California Institute of Technology
Research focus: Molecular microbial ecology of anaerobic communities involved in carbon and sulfur cycling; application and development of combined molecular and isotopic methods for relating uncultured microorganisms to biogeochemical processes and understanding interspecies interactions.

Tracy Palmer University of Dundee, Dundee Twitter: @proftracypalmer
Research focus: Transport of proteins by the twin arginine protein transport pathway, which is found in the cytoplasmic membranes of most bacteria, and the thylakoidmembranes of plant chloroplasts.

Christa Schleper University of Vienna, Austria
Research focus: Metabolic and genomic studies of ammonia oxidizing Archaea from soil, meta-transcriptomics to study the function and structure of complex microbial communities,  Sulfolobus, a model archaeon for studying the origin of eukaryotes and the development of their information processing systems, sponges as nutrient sources and sinks in the marine ecosystem.. and much more.

Young Investigators (Assistant Professors):

Mya Breitbart University of South Florida
Research focus: Diversity, distribution, and ecological roles of viruses and bacteria in a wide range of environments – including seawater, animals, plants, insects, zooplankton, coral reefs, stromatolites, and reclaimed water using molecular techniques (metagenomic sequencing).

Jen Biddle University of Delaware, Twitter: @subsurface_life
Research focus: Genomics and metagenomics of uncultured groups of Archaea, exploring new subsurface habitats, finding a deep biosphere analog in Delaware, microbialites in Pavilion Lake and who or what is responsible for morphology changes in the superstructure.

Kristen DeAngelis University of Massachusetts, Twitter: @kristenobacter
Research focus: Effects that climate change has on soil microbial communities, and applying results towards improvement of next generation biofuels. Examination of  soil carbon dynamics, with a focus on microbial carbon storage and greenhouse gas emissions in the rhizosphere and within the context of plant-microbial interactions.

Kirsten Hofmockel Iowa State University, Twitter: @KHofmockel
Research focus: How plant-microbe interactions mediate ecosystem-specific responses to global climate change. This research connects microbial processes to ecosystem functions to yield new insights into microbial ecology and elemental cycling.

Julie Huber The Marine Biological Laboratory, Woods Hole @JulesDeep
Research focus: The ecology of bacteria and archaea in the deep sea, especially at underwater volcanoes and marine microbial ecosystems of all types, from coral reefs to marine sediments, and the methods and approaches that unite microbial scientists.  Technology development for deep-sea exploration and in-situ experimentation.

Olivia Mason Florida State University, Twitter: @OUMasonlab
Research focus: Understanding how microbial communities respond to perturbations in the environment, such as the Deepwater Horizon oil spill, and in understanding the impact of this response in terms of biogeochemical cycling and ecosystem function.

Rachel Whittaker University of Illinois at Urbana-Champaign
Research focus: Evolutionary ecology of microbial populations,  understanding how the interactions between basic population genetic parameters (mutation, selection, recombination and genetic exchange, neutral genetic drift, and biogeography) shape diversity, promote ecological differentiation, and lead to speciation in the microbial world, how population structures reflect the unique biology and ecology of organisms in the Archaeal domain.

Biotech:
Mimi Healy:  You may not know Mimi Healy, however, when it comes to women microbiologists in biotech, she is one impressive lady. She was CEO of a company called Bacterial Barcodes where her team developed the Rep-PCR technique into a diagnostic test for bacterial identification.  She successfully sold the company to a major microbiology diagnostics leader, BioMerieux, several years ago. Now she is CEO of a company called LaserGen in Houston.  As a woman in science and business, to me, she is an example of how far women can go in science outside the academic path. To the top!

Many thanks all for your contributions.  When I am at ASM this year, I’ll be on the look out to attend talks by the women on this list.  Young women in microbiology and ecology (and us not so young women too) have some very excellent role models!!

I have to pause for a minute to pay homage to and tell you about the humbling power of Palmer Station.  There are only about 30 people that live there in the summer months (Sept-April) but all of them are responsible for running not only a station, but a home.  There are cooks, seamstresses, metal workers, electricians, doctors, scientists, etc. many of them acting in multiple roles on a daily basis.  Each individual plays an integral part to make the station a success even if it means scrubbing someone else’s bathroom floor after lunch.

During turnaround, I was invited on a “boondoggle”, or mini outdoor excursion.  Before any boating activities, one must be prepped for a turn of events, we are in fact in Antarctica where extreme weather can change at the drop of a hat.   All the necessities for survival are brought along, clothing, waterproof gear, food, water,  radios, oh, and don’t forget to sign out on the white board before you go, the station wants to keep track of everyone.  There is a strategic safety plan set in place including 60 gal barrels placed throughout a 5 mile radius stuffed with survival gear just in case…

Six of us piled in to the zodiac and pushed through ice chunks back and forth to Torgerson Island where a colony of Adelie penguins are diminishing due to the changing environment.  The short of it is that loss of sea ice = loss of Antarctic krill, the principle food source for the penguins.   On the way, we passed the over-turned Bahía Paraíso, an Argentine Naval supply ship that sunk in 1989 spilling diesel fuel.  You can only see the very bottom of the hull.  It totally looks like a whale surfacing from far away.

We pulled up to Torgerson and spent about 2 hours checking out the Adelies.  There are a few Chinstrap and Gentoo (picture left) penguins lingering about the Adelie colony as well.  Basically, I followed them from the water where they jump in and out with what appears to be playful glee to their nests full of chicks where mom or dad are keenly watching over their babies who are bustled underneath them.  The chatter amongst them reminds me of neighbors with too much time on their hands starting to call out territory wars.  It seems that even this far South, things don’t change.  This has got to be one of the coolest places on earth, penguin watching in Antarctica, yes!

Next we toured the local icebergs in the zodiac.  These monstrous bohemians are just that, different and unique, no two alike.  As you get closer, you can see the intricate markings on each of them representing thousands of years of ice formation.  It is simple to sense which ones have recently split off the glacier because of the jagged edged ice opposed to smooth curves that seawater has slowly leveled.  I just happened to be looking at the glacier about a nautical mile away when boom!  I saw a calving of the ice (chunks breaking off into the sea from the glacier).  I thought “am I in a National Geographic film?”

Since then, we have left Palmer Station for our 28-day expedition on the ARSV Laurence M. Gould.  The hardcore science has begun.  I am still getting used to my shift of 4am-4pm, not to mention that I go to sleep when the sun seems to be at it’s brightest.  What really has me going right now is all the new scientific knowledge I am sucking up, it’s a different world down here, time to start thinking in new ways.

Cheers!