Scientific Sleuthing

I’ve always had a soft spot for detective novels. I even fancy myself a detective. Not like the ones you read about in pulp fiction made famous by Daly, Hamlet, and Spillane (to name a few), but a sleuth no less. I have spent the last couple of weeks playing detective in lab, you see. I am out on a hunt for molecules produced by these microbes I am studying. This type of investigative work is what attracted me to chemistry and biochemistry. At hand I have a set of known microbial metabolic pathways, a pretty good idea of what some of the intermediate and products are, a lot of chemistry knowledge, and a whole slew of really cool high tech tools.

The method is pretty straightforward. I feed my favorite microbe a known carbon source; it helps if I can radio-label a carbon or another atom on the substrate. I wait for a defined period of time, collect the media, and start the analysis. Step one: spin down cells, take off the supernatant, extract with dichloromethane, …

Yes, it is quite tedious, but many steps later, I have a small sample with metabolites extracted from the experimental microbial growth sample. Now the fun begins. First, what technique do I start with? The simplest is ultraviolet/visible (UV/vis) light spectra. What does the absorption spectra of your samples look like? Can you see something that resembles a conjugated backbone? How about an aromatic ring? Hints of structure here and there.  But then you ask, does the sample contain one or many kinds of molecules? Can I somehow separate these molecules?

In this next step I might choose liquid chromatography (LC) or gas chromatography (GC). Which technique I use depends on certain properties of the molecules I am looking for, are they water-soluble? Are they volatile? For water-soluble molecules I select LC and for volatile molecules I select GC. Both of these separation techniques have a mass spectrometry instrument (MS) attached, allowing me to get a sense of the molecular mass of each separated compound. I inject and watch the molecules fly. Every peak I see reveals a wealth of information about the compounds ‘vitals’. What is the mass of the molecule? Does it fractionate into smaller defined compounds with a known mass? Do the fractionation patters I see match a compound previously characterized?

Next, I isolate and purify the compound using LC, collecting the fractions that have my compound of interest and use Nuclear Magnetic Resonance (NMR) spectroscopy to try to resolve ambiguities due to configuration around enantiomeric carbon atoms. I use proton NMR and carbon NMR and study the chemical shifts I see in my purified sample. From these set of data, I can deduce whether I have a ‘R’ or ‘S’ configuration around my enantiomeric carbon and a host of other structural relationships.

In the end, I have a larges set of ‘clues’ from which I will build a chemical structure of the compounds isolated from the microbial growth media. But that is not enough. In order to stake a claim to a new molecule and possibly a whole new molecular pathway, I have to present my findings to other chemists and microbiologists, convincing them that I have done my sleuthing well. It is very tedious work and most of the time you do not find a new molecule, but every once in a while, you isolate something, a molecule never seen before. The thrill of finding what has not been observed before, drives me to explore the secret metabolic pathways hidden in these microbial species.

Stay tuned to see how this tale turns out. HHH

Thankful for being able to do what it is that I do …

This is what four days in lab looks like.
This is what four days in lab looks like.

It is 7:24 on the day before Thanksgiving. AC/DC is cranked up so loud my ears are starting to bleed and I have at least four more hours to go before I go home tonight.  There are papers and three lab notebooks strewn all over my bench top.  This is my fourth 18-hour day in a row.  A crazy look in my eyes and a five-day growth on my face.  A marathon set of experiments trying to decipher the growth curve of the community of deep earth microbes that I am trying to identify and characterize.

I started this morning over thirteen hours ago by coming in, turning on the gas to exchange the atmosphere in the anaerobic tent loading chamber, and promptly blowing out the seals in the CO2 regulator.  You know that when this happens at 6:30 in the morning, it is not going to be a good day.  I couldn’t find another one in the building (nor in friends labs in a couple of other buildings) so I settled in and waited until AirGas opened later that morning.  Needless to say, it was closer to 1:00 in the afternoon before I got the tent back operational, putting me at least five hours behind schedule for an already jam packed day.  So this is how I find myself with one more hour to wait while my microbial cells sit in the first incubation of many in a long protocol with which I will fix them in paraformaldehyde for later DAPI stain and FISH analysis.

Why do I do this? Why do I put in the long hours crazy hours?  Because I love it.  Plain and simple, that is the only answer I can give.   I delve into the unseen, the unreal, the unknown world of extreme microbes, and try to make it visible, real, solving the mystery of who lives where and how the hell they do what they do in those most inhospitable places in which they thrive.  I have to design an experiment with the full understanding that the equipment does not exist for me to do these experiments.  I cannot go to a shelf and just pick up one of these and three of those and have some technician come over and set it all up for me.  This is truly science driven by your capacity to design, invent, and assemble the equipment that you will need, while doing the experiments at the same time.

Sometimes I forget how incredibly crazy and out of this world what I do is.  I get reminded of this when I try to explain what it is that I do to friends and family.  I get this look of fear and awe when I explain that the things I study grow at 100-200 atmospheres, at temperatures between 50-75 degreed centigrade, and under acidic conditions so extreme it would peel the skin off your bones.  Like I said, this is pretty cool stuff.

Well, I got to go now, the next cell wash and incubation is about to begin.  While all this is extremely exciting, getting to that final answer is laborious, painstaking, and tedious.  Still, I would not trade this life for any other one.  This is what I am thankful for on this week.  I get to do what I love (and sometimes get a little frustrated at) every day.  Take care and have a great Thanksgiving.

Of Chefs and Scientists

Last night I was watching Iron Chef America and there was a young aspiring chef pitting her culinary skills against Iron Chef Bobby Flay. I am always amazed at how the Iron Chefs prepare and pair their ingredients for the five courses. I started to ask myself, where do the ideas come from? How do they choose the color and the flavor for each dish? How do you choose the method for preparation and presentation?

When I woke up this morning, these thoughts insisted in occupying my mind. Lily and I spend some time discussing the kind of training and dedication that it took to gain a mastery of the skills necessary to achieve the title of chef. There is only so much that you can learn in a classroom.  The lessons learned in the o are then performed and perfected through practice and patience.  I remember my short time as a line cook and of the skill that was needed to keep track of the orders, to assure that the food was well cooked, and to make sure that a whole ticket came up at the same time, no matter how many things were on the ticket. I was really good on the line, but I did not come close to having the skill set or knowing the techniques that separate the good from the great.  I learned very valuable skills for food preparation and  presentation, but I never had the chance to apprentice, to follow a master and learn the secrets of great food design.

I have been extremely fortunate to have had a very different experience with my scientific training.  I have received some of the best academic training one could ho.  The interactions I experienced with my fellow graduate students and with the professors at MIT have honed my analytical and observational skills.  Working in the laboratory and developing the methods that allowed me to look at scientific questions using the best and latest tools gave me the confidence and the courage to branch out and try new research topics and fields.  Life as a postdoctoral associate is furthering my set of tools and tricks.

The real test of a chef’s skills are not on the line, but in how they can assemble ideas, ingredients, and skill in their head to give us dishes that inspire and complement our love for life.  Just like a chef, a true test of a scientist’s skills are not at the bench, but in how they can harness a hypothesis and through their skill, design well throughout experiments that increase our understanding and awe of the world around us.

Life under high pressure – the setup

High Pressure Chamber PartsIt was only recently that we began to understand the capacity of microbes to populate the most inhospitable places on earth.  From the inside of nuclear reactors to highly corrosive sulfur environments, microbes are there.   We have found them in 100,000 year old ice cores and in the bottom of the deepest ocean. Microbes were among the first life forms and many of them have evolved in extreme, isolated environments.

One of my research interests is to examine how microbes thrive in these extreme environments.  One unique place where microbes live is deep in the earth’s surface.  Here, they live at elevated temperatures and pressures.   They also have to make do with very little source of nutrients.  Between the extreme environment, low nutrients,  and evolutionary isolation, these life forms harbor the possibility of novel energy regulation and utilization pathways.

To study these microbes, we have to design and build our own experimental equipment.  Currenlty, I am building a high pressure microbial growth chamber for one of the projects I am work on.  This system is going to be able to withstand pressures greater than 160 ATM (1 ATM = 14.7 psi).  We also will have an optical setup with a view cell which will let us examine how our cultures are behaving under these high pressures.  I hope to have the safety cage built this week (the picture is of me drilling the 3/8″ steel plate for the bottom of the cage) and start piping the growth chamber next week.  It is really exciting to see how all of this is coming together.  Stay tuned to see the progress of the high pressure growth chamber.

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