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Team:DTU-Denmark-2/results/Proofofconcept/fungi
From 2011.igem.org
Proof of concept - Fungi
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Filamentous FungiFungi are a diverse group of organisms, whose biological activities affect our daily life in many ways. The filamentous fungi particularly are of great importance in production of medicine, in the industry, in agriculture, and in basic biological research. Some of the filamentous fungal species are pathogenic to humans, whereas others have great value in the production of antibiotics such as penicillin. The fungi are therefore importance for industrial as well as medical production. Filamentous fungi produce a diverse array of secondary metabolites, which serve the pharmaceutical sciences as prolific source of chemical compounds for the development of new drugs. The well characterised filamentous fungi Aspergillus nidulans is a widely recognized model organism. Using the genetic engineering tools available for A.nidulans, serve as a model for investigating polyketides in general.Proof of conceptSeveral separate devices were conducted with Plug ‘n’ Play assembly to verify if the system will work in fungi as well as in mammalian cells. The idea was to target the fungi with a fluorescence protein to specific compartments and in general to express the fluorescence proteins. All the devices have the same promoter, terminator and marker cassette, though with different gene of interest and were all transformed in laboratory Aspergillus nidulans stain: argB2, pyrG89, veA1 by random non-homologues-end-joining integration. We succeed in proving that Plug ‘n’ Play assembly easily transformed and expressed in fungi as we succeed in tagging A. nidulans with two fluorescence proteins. The transformants with integration of our 4 devices change morphology compared with the wild type control strain since the DNA were randomly intergraded and have inflicted some pathways in the fungi. The results are displayed in the following section. Device BBa_K678060The results shows fluorescence spread evenly in the hyphae which correlate with that in device BBa_K678060, the gene of interest, only consists of a green fluorescence protein GFP with no specific target.
Device BBa_K678061The results shows fluorescence spread evenly in the hyphae and with spots of fluorescence. Device BBa_K678061 consists of GFP with a target signal to the peroxisomes which can explain the spots compared to the results from device BBa_K678060. We cannot conclude that the signal is accumulated in the peroxisomes since they are not dyed, though can it be concluded that the GFP signal is target a specific place and accumulated somewhere in the fungi compared to the results from device BBa_K678060.
Device BBa_K678062The results shows fluorescence spread evenly in the hyphae which correlate with that in device BBa_K678061, the gene of interest, only consists of a green fluorescence protein RFP with no specific target.
Device BBa_K678063The results show spots of fluorescence as in Device BBa_K678061 . Device BBa_K678063 consists of RFP with a target signal to the nucleus which can explain the spots compared to the results from device BBa_K678060 and the spores with a single nuclei contains a lot of RFP signal. We cannot conclude that the signal is accumulated in the nucleus since they are not dyed, though can it be concluded that the RFP signal is target a specific place and accumulated somewhere.
Control strainThe control strain shows no auto-fluorescence.
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