Team:SZTA Szeged HU/Results

Results

a) Our main aim was to create a bioindicator E. coli which succesfully detects and forecast the starting of the production of microcystin toxin in the freshwater cyanobacteria Microcystis aeruginosa.
b) We started a succesful cooperation with those roleplayers in our country, who are concerned by algal blooming and the toxin production of cyanobacteria.
c) We succesfully designed and created four composite biobricks (BBa_K3229301-304 named: 'A'-'D') for detecting toxin-related enzyme-production and succesfully transformed four E.coli strains with these biobricks.
d) The Biobricks A’s and B’s final scores suggest that the older is the algal culture the smaller is the tempereature’s effect on lysate - medium difference (see the Model page for the details).
e) The above conclusion seems to strengthen our hypothesis, that the toxin production signal synthesis starts in the cytosol of the algal cells, than it is exported to the environment. The export seems to be accelerated with the increasing population density.
f) The results confirms that Biobricks 'A' and 'B' are sufficient for detecting the starting point of the toxin production.
g) It became clear that the Biobrick C and D didn’t provided sufficient difference between the algal medium’s and the algal cell lysate effect on their transcription. Compared with the low raw values of fluorescence intensity it is probably mean that these Biobricks are not appropriate to detect microcystin production.
h) We succesfully amplified by PCR the variable sequence of the Hungarian subspecies of Microcystis aeruginosa BGSD 243. Our primer design also confirms the hypothesis, that the toxin-related enzyme coding sequences are conservative. The amplified seuquence of the putative promoter will be sequenced in some days. Based on the sequence we create a raw phylogenetic map of the formerly described strains of M. aeruginosa and will find the closest relatives of the Hungarian strain.

Based on our calculations (see Model page) we came to the conclusion that microcystin toxin stays inside Microcystis cells and the synthesis enhancer function is more significant at the Microcystis cultures which were bloomed at 24 °C. On the table below the positive numbers represent the Microcystis cultures and constructs that confirm the above thesis. We can see that the statement is entirely true for the constructs A and B, moreover, the difference between the transcription enhancer function of Microcystis media and lysation decreases if the blooming is at a later stage. This can probably happen because this enhancer needs a minimal concentration inside the cell in order to be transported outside. It approaches this concentration more while the time passes. At construct C and D we can see that this thesis is only partially true. The reason can be that the bidirectional transcription does not work properly within these constructs, or the composite GFP was not able to bond together to form a functional GFP.

In the future, we would like to continue the project and put the icing on the cake. After we will get the sequences of our strain we would like to design a plasmid with the promoter sequence of Microcystis aeruginosa BGSD-243. This plasmid consists of the insert parts with our promoter sequence cloned into a shuttle vector, which can replicate not only in E. coli but in Microcystis sp. as well. Meanwhile, we will try to make a phylogenetic table to place our strain on the relationship with the Microcystis aeruginosa strains, we have already known. As a result, we can make transformed Microcystis and use them to make a more accurate device.