The past 8 weeks

Something I’ve learned this summer is that things always take longer than expected (at least in the case of this project). For the past 8 or so weeks, I’ve been working designing primers that will successfully amplify the genes we want to study, something that I had originally planned to finish by week 4 at latest. Though initially this was pretty stressful and a little frustrating, it’s given me the opportunity to troubleshoot by changing steps of the procedure a little at a time to pinpoint what went wrong.

If you’re interested in more details on what I’ve learned about primer design, read on… 

How do primers work?

Primers are used in PCR, a process that amplifies a certain gene. During PCR, the two strands that make up DNA are melted so that they come apart. Primers are short strands of nucleotides that bind to a single strand of DNA. Both a forward and reverse primer are used, one binding to each of the two DNA strands, so that the region you want to amplify is bounded by primers. This allows an enzyme (Taq Polymerase) to lengthen the primer into a full strand, creating a complete, double-stranded segment of DNA, which is your PCR product.

Source: http://www.science-explained.com/theory/pcr-polymerase-chain-reaction/

The difficulty with Carbonic Anhydrase primers:

Our project aims to amplify genes coding for Carbonic Anhydrase (CA) enzymes. However, CA genes are found in many different organisms (from bacteria to humans!), and even though they all code for the same type of protein, the proteins and the genes themselves are very diverse. So if we could look at all the CA from all the different microorganisms in soil (which is what we want to do, but can’t as of now), the genes would all look very different. This makes it difficult (actually, probably impossible) to find a region that’s common among all CA genes where the primers can bind to. 

The solution:

We ended up designing primers for a very specific type of CA, called Cosase by the researchers who discovered it (Ogawa et al., 2013). They predicted that Cosase was responsible for degrading of COS, so we want to see if there’s enough Cosase in our soils for it to feasibly be responsible for all or most of the COS uptake exhibited by our soil samples. There are relatively few Cosase genes in the NCBI database of sequenced genomes (the organisms whose genetic makeup is completely known), so it will be interesting if we find new, previously unknown Cosase genes!

International Year of Soils

What better summer to do soil research than in the summer of 2015, the UN-declared international year of soils?

http://www.fao.org/soils-2015/about/en/

My Visit to SLAC

Yesterday I got the chance to visit the SLAC National Accelerator Laboratory on Stanford’s campus for the first time! It’s owned by the Department of Energy and operated by Stanford, and it’s the longest linear particle accelerator in the world. To be honest, I don’t know much about what it does except that it speeds electrons up to near-light speed.

Kristin Boye, a postdoc who’s helping Laura with some of the soil analyses, offered to show me what she’s doing there with our soil samples. If you’re wondering what soil has to do with particle acceleration, so did I! I learned that she was working at the Stanford Synchrotron Radiation Lightsource (SSRL) to measure the sulfur content of soil samples. Electrons are diverted from the main accelerator to the circular SSRL facility where they produce the bright X-rays used by the facility. X-ray beams are fed into different stations around the facility where they’re used in all sorts of research, from measuring rock mineral structure to protein structure. Kristin was using the x-rays to shoot at our soil samples, which excites electrons to jump out of their shells momentarily. As they fall back into their original energy level, the electrons emit an amount of fluorescence specific to the atom’s element and oxidation state, so by measuring the emitted fluorescence, she can determine how much of each type of sulfur (sulfide, sulfate, organic sulfur etc.) is present.

Kristin was working at one station, but she showed me around the rest of SSRL. The facility is sunken into the ground, so it felt like walking around a circular basement stuffed with cubicles, compressed gas tanks, and a whole bunch of machines whose purposes I don’t really understand.

She explained to me that using SLAC is free, but scientists who use it have to submit proposals explaining the project they’re working on and why using SLAC facilities is necessary. Then, committees of other scientists review the proposals and give them scores. In the end, the proposals with the highest scores get first priority for picking which equipment they want to use and how long they use them for.

The security on the way in suggested there might be some crazy top-secret research going on (they stopped the bus at a security check point and checked everyone on board for ID), but luckily I was allowed to take some photos!

Experiment Overview

Before I write any posts going to in-depth into what I’ve been doing from day to day, I should probably give an overview of what our experiment is and how it will help answer our research question.

The question we’re trying to answer is which class of the enzyme carbonic anhydrase is responsible for digesting the gas COS.

In Short:

In order to test our question, we’ll use soil samples from around the US (and two from Cambodia!). We’ll measure our soils’ ability to absorb COS gas. Then we’ll see how much the soil microorganisms express the genes which code for the different carbonic anhydrase classes. A positive correlation between the ability to absorb COS and expression of carbonic anhydrase genes would suggest that that carbonic anhydrase class plays a part in COS uptake.

In (Lots) More Detail:

Soil Samples: Since soils have a wide diversity of microorganism communities and chemical and physical properties, we got 20 soil samples from different locations and climates to represent this diversity. To prepare the soils, we sieve them through a 2mm sieve to make sure the particle sizes are more or less uniform.

Gas Measurements of soils: We measure these soils in a quantum cascasde laser spectrophotometer, an instrument which measures the concentration of COS in air based on the specific wavelengths that COS absorbs. To use the instrument, we put the soil sample in the chamber, and mix of air (which has in it an amount of COS naturally found in the atmosphere) is pumped into the chamber. The air is first measured without passing through the soil chamber to see how much COS there is originally. Then, the air is flowed through the chamber where it interacts with the soil, then when it flows out of the chamber, it is measured again to see the change in COS concentration.

Air is run through the soil chamber and measured on its way out to see the change in COS concentration before and after it interacts with the soil.

Measuring Gene Expression: To assess the activity of carbonic anhydrase enzymes in soil microorganisms, we’ll see how much DNA the microorganisms have that code for carbonic anhydrase and also measure how much RNA there is to see how much the carbonic anhydrase genes are being expressed. To do this, we’ll conduct quantitative polymerase chain reaction (qPCR) which allows us to replicate the genes and quantify how much of the DNA or RNA is present.

The first step in doing PCR is designing primers that are specific to the segments of DNA we want to replicate. We’ll create primers for each of the following: all carbonic anhydrase, alpha carbonic anhydrase, beta carbonic anhydrase, gamma carbonic anhydrase, and beta-D carbonic anhydrase. We’re doing this because, although our hypothesis is that the beta-D carbonic anhydrase is responsible for COS uptake, we also want to know what other classes of carbonic anhydrase are being expressed in our soils since its possible that other classes are also responsible for COS uptake.

Once we have the primers, we’ll extract DNA and RNA from the soil samples and conduct qPCR and find out which soils are expressing which carbonic anhydrases!

Working with Isolates: So far in this description of the experiments, I’ve been explaining what we’ll do with the whole soil sample. However, we’ll also work with isolates, which are just a single type of bacteria isolated from anything else. We’ll obtain an isolate from a chosen soil sample and make sure that this isolate has a known genome so that we know exactly which classes of carbonic anhydrase it contains. (We’ll choose one that has the beta-D carbonic anhydrase.) Then, we’ll measure this isolate in the QCL spectrophotometer (the COS-measuring instrument) with COS-filled air to make sure it absorbs COS. Then, we’ll also incubate the isolate in air purified of COS. For these two samples, we’ll then use qPCR to measure the expression of the carbonic anhydrase genes. We expect to see expression of the carbonic anhydrase genes in the COS-exposed isolate but not in the isolate exposed to COS-free air, which would suggest that carbonic anhydrase genes are expressed specifically in response to the presence of COS.

Other soil properties: In addition to the microorganism activity present, soils also have many other properties which we’ll measure in order to try to find a correlation between these soil properties and COS uptake ability. These include: pH, density, water holding capacity (how much water the soil holds when its completely saturated), nutrient content (carbon, nitrogen, sulfur), and texture.

Explaining the Titles, and The Question and Hypothesis

Why would I want to spend my summer studying “Bugs Eating Gas in Soil”, and what does that blog title even mean? Maybe even more confusing is the official title of the project: “Linking Carbonic Anhydrase classes to COS Uptake in Soil Microorganisms”.

In “The Big Picture” post, I explained the “gas” part; the gas is carbonyl sulfide, or COS, which is structurally similar to CO2 and may be a good CO2 tracer.

The “bugs” are microorganisms in the soil. There’s evidence that they decompose COS from the atmosphere using an enzyme called carbonic anhydrase (Kesselmeier et al. 1999) which catalyzes the reaction COS + H₂O  → CO₂ + H₂S. There are many different classes of carbonic anhydrase. The ones most commonly found in soil microorganisms are the alpha, beta, and gamma classes, and we currently don’t know which classes are responsible for breaking down COS.

The question: Which class(es) of carbonic anhydrase is responsible for breaking down COS in soil microorganisms?

Alongside this, we also want to see if other factors (such as soil moisture, soil pH, soil density) correlate with the soils’ ability to take up COS.

The hypothesis: We predict that a sub-class of the beta carbonic anhydrase, the beta-D carbonic anhydrase, is reponsible for breaking down COS. This is based on research by Ogawa et al. 2013 which found that an enzyme (which they called COSase), which is very similar to the beta-D carbonic anhydrase, takes up COS.

References:

Kesselmeier, J., Teusch, N., and Kuhn, U. (1999). Controlling variables for the uptake of atmospheric carbonyl sulfide by soil. Journal of Geophysical Research, 104 (9), 11,577 – 11584.

Ogawa, Takahiro et al., 2013: Carbonyl Sulfide Hydrolase from Thiobacillus thioparus Strain THI115 Is One of the β -Carbonic Anhydrase Family Enzymes. Journal of the American Chemical Society, 135, 3818−3825

The Big Picture

So why is this project important?

When learning about research projects, I think this is the question that interests me the most. As I’ve realized these past couple days working in the lab, research from day to day involves many little tasks (in my case, transferring soil samples between all sorts of containers and tubes). A lot of what makes the little tasks interesting is what they add up to; the procedures add up to experiments which add up to a research project, which is likely just one part of answering an even bigger question.

So since it will give everything else context, I’ll start out with explaining the big picture of this summer’s project.

The Short Version:

To understand and adapt to climate change, it’s important to understand how carbon dioxide in the atmosphere interacts with ecosystems. However, currently, methods of measuring how much CO₂ is absorbed into an ecosystem versus how much is released out are not very accurate. That’s where the molecule carbonyl sulfide (COS) comes in. Since COS interacts with ecosystems similarly to CO₂, it may be a useful carbon cycle “tracer”. In other words, by understanding how COS acts in a given ecosystem, we may be able to predict how CO2 is acting in that ecosystem. Our research looks specifically at how COS interacts with a specific part of ecosystems: the soil microorganism communities.

In More Detail:

To understand and adapt to climate change, it’s important to understand how carbon dioxide in the atmosphere interacts with ecosystems. CO2 can be absorbed into an ecosystem through photosynthesis or release from the ecosystem through respiration. The difference between these to decides whether an ecosystem is a CO2 sink (absorbs CO2 overall) or a CO2 source (releases CO2 overall). However, current methods of measuring this difference between photosynthesis and respiration are not accurate, and we have no reliable way individually measuring these two processes.

That’s where the molecule carbonyl sulfide (COS) comes in. It is structurally similar to carbon dioxide; it just has a sulfur atom instead of one of the oxygen atoms. Because of this, it is absorbed into plants at a rate similar to that of CO2. Unlike CO2, COS is not respired back out of plants. Therefore, by measuring COS activity in an ecosystem and using these measurements in models that associate certain levels of COS uptake with certain levels of CO2 uptake, we could potentially isolate the photosynthesis portion of the carbon dioxide cycle. This would allow us to know precisely how much CO2 is being absorbed into the atmosphere, independent of how much is being released.

However, for COS to be a reliable tracer, we must understand factors that influence its uptake into and release from an ecosystem. It’s currently not well understood how COS interacts with soil. There’s evidence that microorganisms in soil break down COS (absorb it), but we don’t understand what factors influence how much COS soil takes up. This is the question that our research addresses.

We know that COS is absorbed into plants at a rate similar to that of CO2 and that COS is not released back into the atmosphere. However, we don’t yet understand how COS interacts with soils in an ecosystem.

By the end of this project, we’re hoping to be able to look at the characteristics of a given soil and predict how it interacts with COS in the atmosphere, and this information, ultimately, would hopefully be used in carbon cycle models to better understand how CO2 is cycled in ecosystems.