Team:SZTA Szeged HU/Description
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Our main objective was to develop an efficient way to follow the toxin production during water blooming at an early stage. We would like to detect the starting point of microcystin production of blue-green algae namely the toxic Microcystis sp. population. We planned to reach this goal by transforming both Escherichia coli and Microcystis aeruginosa (BGSD 243). We would like to create a biosensor which can detect the starting point of microcystin toxin production via green fluorescent protein fluorescence.
First of all, we made contact with people who are facing to this problem. We met fishfarm owners, surface water agencies and fresh water ecologists- Several researchers told us that the main problem of water blooming is caused mainly by Microcystis aeruginosa, which is a prokaryotic blue green alga species. We learned from these researchers, that the algae start blooming when certain circumstances such as light, temperature, nutrients are adequate. In Hungary the most important factor is the temperature: when water temperature rises above 24 degrees for some days, these bacteria increase their density very aggressively. Meanwhile many of them is going to produce toxins, especially microcystin.
Microcystis aeruginosa produces several toxins, however microcystin which is a cyclic heptapeptide hepatotoxin plays the biggest role in fish extinction.Nowadays the toxin causes more and more deadly infections among animals. Microcystin is synthesized via a non-conventional pathway by 10 enzymes coded by a gene cluster. The regulation of the gene cluster is not clear yet, but probably can be influenced by parameters including trace metals, nitrogen, phosphorus, temperature growth, light, pH and toxin concentration. We tried to measure the effect of temperature.
Just to reiterate, microcystin is synthesized non-ribosomally by a specific gene cluster comprises 10 genes. The promoter of these genes can start transcription in two directions: mcyABC and mcyDEFGHIJ. These proteins are responsible for toxin production. The putative promoter sequence was a necessity to make our constructs. As a result, we searched for this particular sequence in a DNA database, called Ensemble Bacteria and we noticed that its putative promoter is variable between the strains of Microcystis aeruginosa. Due to this, we made an alignment in Geneious Prime to see how should the consensus sequence look like from the promoter of nine different Microcystis aeruginosa and we got that the most similar to the consensus sequence is the Microcystis aeruginosa NIES-843, therefore we asked IDT to synthesize our Biobricks based on this sequence.
We have designed four biobricks each appropriate to predict the transcription of the genes responsible for microcystin synthesis. Two of them consist of the following parts: at the beginning, there is our promoter sequence where we placed an RBS followed by GFP gene. In addition, there are two types of transcription terminator sequences. Both biobricks contains the same parts, however the putative promoter sequence was reversed in one of them (for genes A,B,C), thus we can see whether the transcription occurs in both directions or not.
The other two consist of the putative promoter and two GFP gene parts in two directions. This GFP only glows when both proteins are synthesized at the same time and linked together, thus we can measure if transcription starts in both directions at the same time. We also placed RBSes (ribosomal binding site) between the promoter and GPFs, and the two types of transcription terminator sequence after GFPs. These two constructs were made with the original promoter sequence, but the order of the two GFPs were changed in the two constructs. Genes from fluorescent proteins will be expressed in the engineered Escherichia coli bacteria and Microcystis aeruginosa.
We asked for a special Hungarian strain from the researchers and amplified the putative promoter from its genome to sequence - hoping that we can clarify the phylogenetic relationship of this strain to others already sequenced which can be an essential help to the people we mentioned formerly at the Human Practices slide.
As we formerly mentioned, the water temperature is a key factor in blooming in our country. Therefore we set up two cultures at two different temperatures and the blooming at higher temperature was obvious (see the photo of the cultures.) We took daily samples from both cultures for 10 days. As we don’t know whether the transcription signal of the putative bidirectional promoter is intra- or extracellular, we used both the liquid medium and the lysated Microcystis algal cells to possibly induce the mRNA generation after treating the transformed E.coli with them. The measurement goal is to determine the difference in GFP fluorescence between pairs of treated and transformed E.coli: one member of the pair was treated with an algal sample cultured at 24 °C and the other with material from the algal culture at 30 °C.
Inspiration
In our project we wanted to deal with an issue on environmental protection that affects our land, Hungary as well. Since we have many important lakes (Lake Balaton, Lake Neusiedl, Lake Velence) where algal bloom can occur, we chose this problem as the topic of our project.
Algal bloom and eutrophication disturb the natural biocenosis of lakes as it leads to the excessive reproduction of algae, causing plant and fish die-offs, and later the siltation of lakes. The consequences of these are the discolouration and the unpleasant smell of water and there will be more and more carcases in the lake. Moreover, due to harmful algae various toxins can accumulate in the water.
In Hungarian economy, lake tourism has a significant role, there is a huge income due to Hungarian and outlandish tourists visiting our lakes. But the aforesaid problems decrease considerably the proceeds. According to a research (from 2018) lake tourism earns 7% of our GDP.
Algal bloom is not just affecting Hungary but is also a global problem in our present days. That’s why we thought that it would be useful to be able to detect algal bloom already in an early state, because then it might be possible to prevent most of the harms.
Description
In our project the team is dealing with algal bloom. Algal bloom is a global problem affecting both natural and artificial standing waters. An algal bloom is the excessive increase of algal population in marine water and freshwater. This process leads to numerous negative and harmful consequences. As the algae cover the water surface, the water transparency decreases significantly and light does not reach the deeper water-layers, which results in the destruction of the aquatic vegetation. This conduces to the depletion of dissolved oxygen, the consequences of which can be considerable fish die-offs and on a longer view the eutrophication and siltation of lakes. Besides there are many algae species which can secrete harmful toxins.
In the picture we can see algal blooming's late estate. There's the green area that appears when the bloom starts. As the time goes by the algae die and they become dark and brown, which is probably éven more worse, because they can't do photosynthesis, although they keep their other harmful features (for example: cut off the light...).
On the photo there's the first signs of algal blooming. The water turns into greenish blue and the algae merge into light green colonies.
We are focusing on a bloom forming, toxic genus of cyanobacteria called Microcystis, more specific on two species of the genus, Microcystis aeruginosa, which is one of the most known and examined species. Microcystis species fundamentally occur in shallow lakes or in the shore-regions of deeper lakes. At nights the colonies sink down to the bottom, but in day-time (or at least in some parts of the day) they come up the surface, sometimes covering it entirely. The advantage of this strategy is that on the bottom there are more nutrients, so the algae absorb them there, and photosynthesize on the surface.
The Microcystis species can produce various toxins, which are detrimental to animal and human health; in the respect of our project the most important is microcystin. Microcystin is a hepatotoxin, and acute cases of microcystin poisoning may cause rapid death. Microcystin is a cyclic heptapeptide, which has around 90 isoforms. Most of them have a common structure cyclo(Adda–D-Glu–Mdha–D-Ala–L-X–D-MeAsp–L-Y). X and Y are variable L amino acids (most frequently leucin and arginin). Microcystin is synthesized nonribosomally via a mixed polyketide synthase/nonribosomal peptide synthetase system called microcystin synthetase. The gene cluster encoding this microcystin sythetase complex consists of 10 genes (mcy genes). The mcyABCDEFGHIJ genes are transcribed as two polycistronic operons, mcyABC and mcyDEFGHIJ, from a central bidirectional promoter between mcyA and mcyD.
These genes produce the toxin's enzymes
Our goal is to develop a system which is able to detect microcystin production, and thereby the toxicity of water. Therefore we constructed several plasmids where mcy genes are replaced with gfp (green fluorescent protein) genes. We are going to transform them into Escherichia coli and Microcystis aeruginosa. By adding them to cultured wild-type M. aeruginosa we expect the gfp genes to be transcribed due to cell-to-cell communication. We are taking samples from the growing cultures in order to determine the algae concentration which they start to produce microcystin at.