journal tabs

Journal
Cara Pekarcik's picture

FREEDOM!

At around 2130 last evening, I noticed that the ship was no longer following the typical pattern of the last six days (reverse/forward/ram/repeat). Instead, we were moving forward – and we were not stopping. After 6+ days in the heavily packed, snow covered ice, we reached a point where the snow and ice combination was small enough to allow us to break through and keep going. I went to the bridge to get a bird's eye view of the ice edge. Without much fanfare, we left the pack ice at 2240. It was a bitter-sweet moment for me. It will most likely be the last time I see the mountains of Antarctica and the pack ice (that's the bitter part). The sweet part has to do with the science. Our departure from the ice allows us to squeeze in one more day of science before we must depart for the Drake Passage and ultimately Chile. The science team has decided on a deep trace metal cast (3500m/10500ft) followed by water collection. I will explain more about this in tomorrow's journal.

Guest Author

Kris Gomes from the University of Rhode Island expressed interest in writing one of the journals. With just a few days left in the research cruise, I thought it might be great if Kris could explain a word that has been tossed around for the past few weeks...metatranscriptomes. I have seen large carboys of water being carted around the ship from deck-board incubators to walk-in coolers, but I really was not sure why these large quantities of water were necessary. Thanks to Kris' explanation, I have a better understanding of this important analysis technique. The following is what Kris had to say about metatranscriptomes.

Deck board incubatorsThe flow-through incubators bring sea water through pumps to the incubators. This allows the bottles to receive ambient (natural) temperature and light during incubation.

Incubation bottle with krillThe 20L carboys remained in the incubators for 12 days. You may notice an Antarctic krill swimming in the incubator. The krill traveled into the system as sea water was pumped from the surroundings through the pipes of the incubator system.

The Central Dogma

Metatranscriptomes are an important tool biologists can use to understand how communities of cells in an environment, such as diatoms and bacteria in the Southern Ocean, respond to their environment and to each other. Before we talk about what metatranscriptomes are its important to understand the relationship between DNA, RNA, and proteins. In a previous journal, we discussed the importance of DNA and RNA molecules and their ability to convey genetic information. This relationship is a part of what is referred to as the central dogma, the foundation of molecular biology, which describes the flow of genetic information from DNA to RNA and finally to the production of proteins.

Central dogma diagramDiagram showing the flow of genetic information from the DNA inside the cell's nucleus to the production of proteins for structural and chemical functions.

In a cell, DNA serves as the blueprint of all the proteins that an organism is capable of expressing (making). During the process of transcription, or RNA synthesis, the genetic code for specific proteins are copied into RNA transcripts, acting as “instructions” telling the cell which proteins should be made. These “instructions” are then read by cell parts called ribosomes and used to produce proteins. This process is referred to as translation. The proteins act as the “functional tools” of the cell and/or the organism. These proteins can act as enzymes (like the lactase that breaks down the lactose sugars in the milk that you have with breakfast), structural proteins (like the keratin protein that makes up your hair and nails), etc. Cells can produce different proteins throughout their lives depending on their role inside the organism and/or changes in their environment.

What Is a Metatranscriptome and What Can It Tell Us?

Metatranscriptomes refer to the pool of all RNA transcripts that are being produced by the organisms within a specific community at a specific time. They can be thought of as genetic snapshots of what a community is doing and how they are responding to their environment. In other words, what RNA (and eventually what proteins) are the cells creating at a given moment in time.

Under changing conditions cells are able to transcribe or copy different RNA transcripts and in different amounts in response to their physiological needs or environmental stresses. This change in the number of specific RNA transcripts produced is referred to as differential expression. As we discussed, RNA serves as the “instructions” to the cell of what proteins should be synthesized. Changes in the amounts and types of RNA transcripts produced result in a corresponding change in protein synthesis. The differential expression of RNA transcripts and the resulting proteins allows for an organism to physiologically change in response to differences in their environment, such as the differing nutrient levels being tested in our incubation experiments.

By sequencing the large pool of transcripts collected from these experiments we are able to read and begin to identify the sequence of RNA transcripts. These sequences give us an idea of the proteins being made by the diatoms, or other studied cells. We can begin to understand how the organisms in the community are responding to the different conditions being tested in our incubation experiments. Put simply: do diatoms produce different proteins in response to the different nutrients in each incubation group? This information allows us to understand not only how single organisms respond to the different conditions being tested but also how the community as a whole responds and interacts with each other.

Sample Collection

Sequencing a metatranscriptome requires a large amount of RNA, which means the science team must filter large quantities of water to collect large amounts of biomass. To get the amount of biomass required, the metatranscriptome experiments are setup in large 20 liter carboys, allowing for a greater number of cells to grow during the course of the experiment. Just like the water from the other incubation experiments the metatranscriptome bottles are filtered down to collect all the biomass. The filters are flash frozen in liquid nitrogen and brought back to lab. Once at the lab the pool of RNA from the cells will be extracted and sent out for sequencing, so that we can begin to make comparisons of how the communities collected respond to the different experimental conditions.

Metatranscriptome fltrationThe filtration set-up for four metatranscriptome sampling bottles (clear carboys on counter). The water from the carboys is pumped through the large filters handing into the carboys on the floor of the cold room. Large quantities of water are filtered for metatranscriptome analysis.

Filter comparisonThe filter on the left is the 25mm (diameter) filter rig used to extract biomass for RNA and DNA sampling. The filter on the right is the larger (47mm diameter) filter rig used for metatranscriptomes. Both house 3 micron filters in order to catch diatoms and other biomass, but one will hold much more than the other.

Comments

Add new comment

Vivian Tran's picture

Vivian Tran said:

Hi Mrs. Pekarcik! I was wondering why you would flash freeze the filters in liquid nitrogen, but maybe I got my answer. Is it because you don't want the cells to change in any way?
Cara Pekarcik's picture

Cara Pekarcik replied:

Hi Vivian - you are correct. The science team does not want the RNA to change in any way. Cells can create different molecules of RNA if the environment changes. If they are not flash frozen and just placed in a freezer in order to eventually freeze, the slow change in temperature and the absence of light may cause different genes to produce different types of RNA. The science team would like to know more about the natural metatranscriptome, instead of one created my stress or change in environment. On 2016-10-11 10:12, PolarTREC wrote:
Shane Mitchell's picture

Shane Mitchell said:

How useful are the metatranscriptomes, and what are the different kinds of stress can you tell the diatoms are in?
Cara Pekarcik's picture

Cara Pekarcik replied:

As Kris explained, the metatranscriptome information is important in helping to understand the population within a given area.  Stress can be related to temperature, light, salinity, etc.   From: PolarTREC <webmaster@polartrec.com> To: <cpekarcik@polartrec.com> Sent: 10/20/2016 7:14 AM Subject: Re: Shane Mitchell commented on 10 October 2016 Metatranscriptomes ((
Marvisa 's picture

Marvisa said:

What did you gather from the large amounts of water? what experiments did you perform on them?
Cara Pekarcik's picture

Cara Pekarcik replied:

Marisa - this is what all of my other journals are about.  Check out some of the journals entitled CTD, Incubation Sampling, Iron Hypothesis, etc. to find out more From: PolarTREC <webmaster@polartrec.com> To: <cpekarcik@polartrec.com> Sent: 10/20/2016 7:23 AM Subject: Re: Marvisa commented on 10 October 2016 Metatranscriptomes (
Gina Ash's picture

Gina Ash said:

Do metatranscriptones functions vary between each organism, for example, would it be the same as a diatom as in a human?
Cara Pekarcik's picture

Cara Pekarcik replied:

Hi Gina - parts of the metatranscriptone could be the same if both diatoms and humans are producing the same RNA molecules.  We will learn (later this spring) that RNA sequences produce amino acid sequences to make proteins.  It is possible that diatoms and humans can have the same gene to produce a protein and that they could be the same within a metatranscriptome.   From: PolarTREC <webmaster@polartrec.com> To: <cpekarcik@polartrec.com> Sent: 10/23/2016 5:44 PM Subject: Re: Gina Ash commented on 10 October 2016 Metatranscriptomes (((
Bryan Keller 's picture

Bryan Keller said:

Hello, I am interested in the taxonomy of the phytoplankton and bacterial communities that your team is studying. Did you use metatransciptomics as the main tool to characterize the biodiversity of these taxa? Were there any other methods used? I read that the extracted RNA has to be sent out for sequencing, but did you notice any qualitative trends with respect to the abundance of phytoplankton? If so, did anything surprise you about the environmental parameters that potentially influenced the observed trends? Thank you for your time and good luck with the rest of your study, Bryan
Cara Pekarcik's picture

Cara Pekarcik replied:

Hi Bryan - thank you for the email. I have forwarded your question to the research team and will post the answer when it is available. From: PolarTREC <webmaster@polartrec.com> To: <cpekarcik@polartrec.com> Sent: 11/7/2016 5:49 AM Subject: Re: Bryan Keller commented on 10 October 2016 Metatranscriptomes
Cara Pekarcik's picture

Cara Pekarcik replied:

Hi Bryan This is Bethany the chief scientist on NBP16-08 responding to your question about how we classify taxonomy and what kinds of data we will get from the metatranscriptome analyses. We are looking at phytoplankton communities in several ways: Optically using a Flow Cam imaging system that captures frame by frame images of cells at each location and molecularly by targeting the 16S rDNA gene (bacteria) and 18S rDNA gene (eukarya and diatoms). The metatranscritome analysis will reflect global suites of genes turned on under different conditions. We can infer taxonomy from these data by comparing their homology across organisms from different taxa. To understand all of these data will take a lot of post-cruise analyses.  The Flow Cam images will need to be analyzed and statistically compared. The sequencing and analyses of sequence data will be done after we extract the nucleic acids back in our labs. Your question regarding the environmental parameters forcing trends in community composition is a great one and one that we will be able to answer when we can use our data from community composition from various locations with other measured environmental parameters with statistical measures that help us define the factors that best explain community composition differences. From: PolarTREC <webmaster@polartrec.com> To: <cpekarcik@polartrec.com> Sent: 11/7/2016 5:49 AM Subject: Re: Bryan Keller commented on 10 October 2016 Metatranscriptomes (