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.