A diazotroph specific microarray

Things have finally settled a bit after the climactic licentiate seminar two weeks ago. Of course life at work goes on and there are plenty of things to do, both scheduled and more surprising events.
For example, my supervisor showed up in my office the other day and asked me to do a seminar in the departmental marine seminar series due to a late cancellation.
Sure thing! It's good practice and I know my presentation well since the licentiate seminar (I practiced almost every day for two weeks), but it will still require a bit of time and preparation.
Hopefully more people will show up than for my licentiate seminar, but I won't get my hopes up.

So besides doing DNA extractions of samples from the Meekong River plume in the South China Sea, I'm closing in on the finalization and first test samples for my 3rd project, the microarray.

So I will explain this project and the approach in a bit more detail.
To start from the beginning, a microarray is basically a genomic tool for identifying and quantifying the expression of particular genes in a cell. This can either be done on the whole genome or a subset of genes or genetic pathways decided by the scientist (me).
The approach is based on RNA in our environmental water samples, or more specifically messenger RNA (mRNA), which codes for proteins within the cell. This mRNA is, prior to addition to the microarray, replaced by fluorescence labeled complementary DNA (cDNA).
The microarray itself comprises of one probe for each gene of interest, which together makes up a probe group, and a probe is a short DNA sequence (in this case ca 60 nucleotide bases) which is designed to bind and hybridize with the labeled cDNA from our samples (if there is a match).
When all of these things are sorted out, the microarray will be printed on a glass slide lined with thousands of tiny wells (16 000 to 128 000 depending on the format). Each well corresponds to one gene and also have the probes to target that gene.

The glass slide at the bottom, scaled up are the thousands of wells and on top are the DNA probe sequences within each well.
Credit: Genetic Science Learning Center. (2013, February 14) DNA Microarray. Retrieved June 06, 2017, from http://learn.genetics.utah.edu/content/labs/microarray/

After the sample (with labeled cDNA) has been added to the microarray, the slide will be scanned by a specialized instrument connected to a computer and an analytic software. The setup will identify and quantify the fluorescence from the wells and subsequently compare samples.
The resulting data (values) will allow me to describe and compare the magnitude of up- and down regulation (expression) of genes using a set of reference genes which should always be expressed.

For my project I'm investigating the gene expressions and also the genetic repertoires (which is not restricted to microarrays) for the heterocystous diazotrophic (nitrogen fixing) cyanobacteria which live in symbiosis with diatoms in the tropical open oceans. These cyanobacteria are three strains of the genus Richelia and one strain of a marine Calothrix (a close relative of Richelia).
The aim is for me, to not only describe the cellular activities and differences therein between these strains, but also try to link them to environmental parameters and the differing hosts.

This is how the annotated (described function) genome looks like. Further to the right are (not in the picture) the nucleotide sequences for each gene (line)

Most of the work has already been done. The bulk of this analysis was digging through all the genomes to find relevant genes and genetic pathways (e.g. stress response, nitrogen fixation, cell division and nutrient uptake). At this point I'm finalizing the probe designs and assembling the microarray, which took me much more time than anticipated, mostly due to cross hybridizing probes, which basically means that they bind to more genes than just the one I intended.
However, that problem is enough for an entire blog post of its own.

Licentiate thesis

The long awaited day of my licentiate thesis defence is closing in (31st of May). Next week I will present my work to this point, at a 30 min seminar followed by a scientific discussion with my opponent, Bethany D. Jenkins from University of Rhode Island, USA.

The content of my thesis (which I also got in print today) is basically a 'kappa', which is an introductory summary of sorts, followed by my first and second paper (the first in review and the second published). The papers were obviously already written prior to the licentiate thesis, so I spent quite some time writing the 'kappa'.
There are certain guidelines for this summary, and is my opportunity to show that I meet the requirements for my examination according to the Swedish Ordinance of Higher Education. As usual, getting started with the writing felt like an uphill run in mid July. It was a struggle in more than one way, but as soon as I reached the summit, and got a better view of where I wanted to go, things started to quickly fall into place as I rushed downhill. With good input from my supervisor I could quickly wrap it up (in difference to my first one year manuscript...).
All in all, it introduces readers to my field of research, including the methods, touch upon the motivation behind the studies, followed by a synthesis of the main findings, and lastly, discussing how to move forward with my research (project no.3, which I will get to in my next blogpost).

The first paper can be found here:

http://www.biogeosciences-discuss.net/bg-2017-63/

This is the results of my first scientific cruise, and it's a lot of science. It covers cyanobacterial nitrogen fixers which are either free-living or lives in symbiosis with another phytoplankton in the Western Tropical South Pacific. In short, we found that there was a clear separation of nitrogen fixers based on a depth-temperature gradient, but not in the way we would have thought. That same gradient has been observed elsewhere. We also found interesting differences between one of the cyanobacteria and its proposed host, which contradicts much of the recent literature.

The second paper can be found here:

http://journal.frontiersin.org/article/10.3389/fmicb.2017.00810/full

I'm really proud of my contribution to this paper, especially since it includes a sophisticated statistical model, something I never thought I would do.
The results of the model were a central part of the paper and an important addition to the research in my field and in particular on the diatom diazotroph associations (DDA) that this paper cover.
The data was generated from two cruises which my supervisor went on outside of the Amazon River in the Atlantic Ocean in 2010-11. We wanted to explain and predict, by use of the piecewise Structural Equation Model (SEM) the Amazon river plume's impact on the DDAs in this region.
In summary, we found turbidity to be the most prominent environmental parameter, but the SEM also allowed us to explain the DDA distributions in relation to the river plume. Lastly, we were able to distinguish between the environmental preferences of two strains of the DDAs, which has never been done before. The results of the model indicated that one is more coastal and the other is more oceanic.

So, there it is. All my hard work of the last two years is now nicely compiled in this thesis. I'm super happy and proud of my achievement so far. During my last year of undergraduate studies I would never have even dreamt of working with either statistics (modelling) or microbiology (because I was terrible at it). Look where life took me. So whoever you are, whatever you're doing, know that with the right mindset, no challenge is ever too great!

A brief summary on 2016

It's about time I update the blog with something of interest, and if nothing else, then a brief summary on the turbulent year of 2016.

When last I wrote in my blog I was just starting to write the manuscript of my first paper. It was not as easy as I would have hoped and in the end it turned out to be year long struggle which felt like it would never end. Subsequently, I lost all my motivation to write anything else during that time.

While struggling to finish the first manuscript I also started working on something totally different in the form of a multivariate statistical model, called Piecewise Structural Equation Model (piecewise SEM) which was developed and applied by Lefcheck in 2016.
The model was something I'd thought about for a while after getting comments on my, comparably simple, statistics done in the first manuscript (which then of course was expanded to include multivariate redundancy analysis (RDA)). It was far outside of my comfort zone, but with the help of Carlo Berg at Science for Life Laboratory in Solna, we put together an R script and code to apply the model on a dataset generated in a previous project (in the Atlantic in 2010-11) which my supervisor participated in.
At first it felt like a longshot and the timeframe was more than a little optimistic since there was a hard deadline for a specific special issue in the scientific journal Frontiers in Microbiology.
However, as our work progressed the optimized model returned more and more satisfactory results and it was decided to be the major part of my second manuscript.

So, while I was struggling with both novel statistics, two manuscripts and a quickly approaching half-time point (where I still had nothing to show), there were internal conflicts that had to be dealt with.
Suffice to say that we're all humans (even researchers) and there are bound to be frictions in a tight, high-achieving workgroup. Still, it was not an easy time, although, I believe we all came out stronger in the end.

By the end of 2016 I started to see the light in the end of the tunnel, and it was thankfully not a train. I was the train, and things were moving fast!
After 36 updated and corrected versions of the first manuscript it was submitted at the same time as the second manuscript on the piecewise SEM, which was written and completed in less than a month.

In hindsight, I've learned A LOT, both about my research and myself as a human being and researcher, during 2016. Workwise I will likely not remember the year of 2016 fondly, but I would not have been where I am now without it.

Stay tuned for an update (in the near future) on the manuscripts and my current, third project.

A long time coming

I know this blog post has been a long time coming, but I still intend to keep it short.
There has been no revolutionary happenings but I've been occupied with a lot of different things both professionally and privately.

The year is nearing its end but the holidays doesn't mean much for my workload. I still have to be productive even though I will of course treat myself with a proper Christmas celebration with my family.

I'm wrapping up the last samples and data points for my first publication this week and already starting on my next project, and suffice to say that both are looking really exciting.
Looking back on the data that I've accumulated, I've now run the qPCR instrument through 10 different microbial targets adding up to ca 1350 samples! Including the environmental metadata it will be a huge dataset.
I will get back to you on my new project soon and hold off with my exciting results until I've gotten it published in a scientific journal.

One year anniversary as a PhD student

Today was officially my one year anniversary as a PhD student. Time has just flown by, although I've gotten a lot done and at the same time had great fun.
Doing my PhD has only during my first year proven to be no easy thing, however, I didn't really expect anything else but hard work. A true test of commitment, resilience and adaptability.
So in spite of that fact and state of mind, the first year has been filled with ups and downs and in the end I had to deal with a lack of communication ending in a shocking revelation that what I did was, timewise, not enough.
To be fair, I didn't know what was required, but I quickly had to realise my shortcomings and reorganise my work time to make it more efficient. I also had to demonstrate more outward commitment and passion regarding my project.
Honestly I didn't find it too hard to manage my time at work, however, it was not without its tradeoffs, including gymtime and family.
Outwardly showing my passion for my project was a somewhat more difficult story though, since I guess I'm just not that kind of person. I think I solved it in an agreeable manner though, by constantly sending updates, questions and suggestions to my supervisor whenever something new came up, sometimes several times a day.
As a complement to my first literature review I have to write down my thoughts, plans and hopes for my project. This is where I'm supposed to find my niche and dig down into something that really excites me. I've always been a generalist in my interest of marine biology, and prior to starting my PhD I thought of myself as a marine ecologist. Pursuing questions at the ecosystem level. I still have that interest, often linked to global environments. That is the major strength of my current project where I study tiny microorganisms with a clear link to biomes and global environmental impacts. I'm not a modeller though, so most of these links are interpretations of data and conclusions drawn from previous and complimentary research to form hypotheses and theories. Nonetheless, it's still plausible and damn interesting. There are no experiments that could ever cover a global scale, with acceptable resolution, anyway.
I'm still fascinated by the prospect of "seeing the future" though. My proposed ocean acidification experiment was shut down largely due to technical difficulties and more experienced researchers reluctance to collaborate on the subject.
I'm tempted to try my luck down the road of global warming instead, which is the other major issue of global environmental change. That research question recently showed a lot more promise when we finally got one of our studied microbes in culture.

Back to "real life". What little spare time I have in Stockholm is filled up by the gym so that's fine, but there is a delicate balance between work and family that every healthy relationship needs to accomplish. Our solution was parental leave on Fridays.
Because of this I'm extra thankful for doing my PhD in Sweden where I, as a student, have the perks of both studying and being employed by the state.
It's still somewhat tedious to commute on a weekly basis. I've done it for so many years previously so I've kind of gotten used to it by now, but never this far. However the distance is not the main issue most of the time. Down-time on the train are usually perfect opportunities for reading, writing and napping. No, the worst are those sudden moments of stabbing longingness for my crazy kids and lovely wife. I don't think that is something that will ever change, but I would argue that this, in the bigger picture, is something positive. I just have to deal with it.
In that regard the gym is my sanctuary. It always has been, and always will be. In there it's just me and the iron. It doesn't matter how passionate I might feel about my work. The gym is always... different.

First manuscript soon to come

Sometimes I just want to scream out the results and significance of my research. These things are too cool not to be communicated. I'm finding and observing trends in my data that both excite and begs for further questions and investigations. This is the beauty if science where you're never truly done, and as a scientist you're driving factor is an ever burning curiosity that can't be put out.

My first manuscript for a publication is drawing near. I'm soon finished with my lab work for this initial project. Then, hopefully, I can scream out to all that want to listen (and a few more) that what I found was actually really cool and that it has some implications for what we currently know about open oceans, as well as raising further questions worth pursuing.
Oh, how I love straight-forward graphs with clear patterns. It makes data analysis so easy when the graphs tell you where in your dataset the statistically relevant trends are.
Or perhaps peer-review (or my supervisor) for my first paper will in no uncertain fashion tell me that I need to calm down.

Motivation - Perspective is everything

The following are a few quotes that I found in motivational gym videos on YouTube.
In my opinion they can be applied on any activity or life event, be it work, a difficult assignment, exercise or just reaching one's individual goals and dreams.
If you're into bodybuilding (even remotely), then I recommend you to check out JerichoDMZ's motivational videos on YouTube.

"Work on your focus, perspective is everything.

The problem with most of you is that you see difficult as something negative.

I want you to see difficult differently. Do you hear me? I need you to see difficult differently!

That weight-lifter when he’s looking at those weights, he got that iron. That iron is heavy, but he isn’t looking at that iron like “look at how heavy that iron is”.

He knows what it’s going to do. It’s going to tear his muscles to pieces. He gets it! What he understands is that if it tears them down they will rebuild themselves. They will rebuild themselves that much stronger!

The more you go through, the more difficult it is, the more challenging it is, the harder it is. Are you hearing me? Don’t quit!

I suggest to you, if you want to become diamond, write down five reasons why it is worth it for you to become a diamond.

To experience that level of achievement, what is it that will give you the drive?

What is it that will ignite the courage in you to get up and come back again and again and again?

If it was easy, everybody would be good at it. If you want something you never had before, you have to be willing to do something you’ve never done before.

This is kind of hard to understand but, sometimes you can try so hard at something. Sometimes you can be so prepared, and still fail.

And every time you fail it’s painful. It causes sadness.

I’ve often said that a man’s character is not judged after his celebrates of victories, but what he does when his back is against the wall.

So no matter how great the setback. How severe the failure. You never give up.

You never give up. You pick yourself up. You brush yourself off. You push forward. You move on. You adapt. You overcome. That is what I believe!"

Go out there and show yourself, and everyone else, what you're made of. Today is your day!