Murray Darling Citizen Scientists

Welcome Citizen Scientists!

The fish kills of 2019 at Menindee, apart from devastating swathes of Australia’s major internal waterway, highlighted the lack of evidence-based decision making and predictive capacity for the catchment managers. In order to better understand and hopefully mitigate the effects of blue-green algal blooms we have instigated a “citizen science” project along the Murray-Darling River system. Our purpose is to investigate the types of cyanobacteria occurring at different times of the year and correlate environmental conditions, like temperature and nutrient levels, with their potential growth rate and toxicity.

After your samples have been received, we will conduct various experiments to determine the nature of these algal blooms. As seen in the flow diagram, the bacterial DNA will be collected from each sample and sequenced using next generation techniques. This will allow us determine the bacterial species present in each bloom. Additionally, the collected DNA will be assessed for the presence of toxin-producing genes. Finally all of this data will be mapped back to the environmental data to provide a holistic representation of the cyanobacterial blooms plaguing Australia. For more information on each technique, see below.

 

DNA Extraction

DNA extraction is a common scientific procedure to isolate the nucleic acids from the nucleus of a cell. It is a simple process that uses both chemical and physical methods to rupture a cell membrane and purify the cell DNA from the cytoplasm while removing all of the additional components like protein and RNA that aren’t desired.

  1. Samples are collected
  2. The cell membrane is broken open to access the DNA
  3. A salt solution is added to clump all of other cell components together (lipids, RNA, protein)
  4. The samples are spun using a centrifuge to separate the debris clump from the DNA
  5. The DNA is purified to remove any remaining protein or salts as well as any chemicals that were added to aid the extraction process
  6. The DNA is now ready for analysis!

DNA Sequencing

Ribosomes are machines within cells that produce protein and are usually found in very high quantities. By targeting a specific gene responsible for making the ribosomes we can identify the species of our sample. For bacteria, we focus on the 16S ribosomal RNA genes (16S rRNA) as they have a very slow rates of evolution and therefore remain constant and unique for each species.

To construct a phylogeny, or bacterial family tree, we sequencing the 16S rRNA by first performing a polymerase chain reaction (PCR) to generate large quantities of the DNA. This DNA may then be sequenced using next generation techniques. All DNA is comprised of four bases; A, G, T, C. When we sequence these, the DNA strand is copied thousands of times! To generate a new DNA strand, fluorescent DNA bases are incorporated into the growing chain. Each of the bases is a different colour and so by recording the unique colour pattern we can interpret the DNA sequence of each bacterial species within a sample.

A more in-depth explanation of 16S rRNA sequencing is included in the video below from Illumina. Alternatively, this paper provides a great overview of the technique.

 

 

Toxin-producing potential

Thankfully, not all cyanobacteria produce toxins- but it is still important to assess their ability to potentially cause harm. There are various ways to assess the potential for toxin production in cyanobacteria. Common methods include gene detection, chemical monitoring or an enzyme-linked immunosorbent assay. The method for detection and monitoring is often chosen depending on the severity of the threat and multiple methods can be used together.

We will begin by assessing whether the samples you collected have the potential to be toxic. While a particular cyanobacteria species may have the genes required to produce toxins, they do not necessarily actively make them! So the first step is to check whether the cyanobacteria even have the genes necessary for production. We will do this by performing real time PCR:

  1. DNA is extracted from the samples
  2. DNA primers are added that target specific toxin producing genes
  3. A fluorescent dye is added (when it is incorporated in the growing DNA strand it will glow brightly)
  4. The Polymerase Chain Reaction is conducted, generating new copies of the target region

We are able to monitor this process as it occurs and therefore quantify the number of genes per cell, and per sample!

 

If you would like to participate and join our citizen scientists, please register you interest here.