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From 2011.igem.org

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  • The Plug 'n' Play
    • Assembly system
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  • Background
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  • Proof of concept
    • Mammalian Cells
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	Protocols 1 Proof of concept in fungi 2 Device BBa_K678060 3 Device BBa_K678061 4 Device BBa_K678062 5 Device BBa_K678063 6 Control	Proof of concept Several 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_K678060 The 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. Aspergillus nidulans with device BBa_K678060 - DIC light. Aspergillus nidulans with device BBa_K678060 - GFP light. Aspergillus nidulans with device BBa_K678060 - Front Aspergillus nidulans with device BBa_K678060 - Back Device BBa_K678061 The 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. Aspergillus nidulans with device BBa_K678061 - DIC light. Aspergillus nidulans with device BBa_K678061- GFP light. Aspergillus nidulans with device BBa_K678061 - Front Aspergillus nidulans with device BBa_K678061 - Back Device BBa_K678062 The 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. Aspergillus nidulans with device BBa_K678062 - DIC light. Aspergillus nidulans with device BBa_K678062 - RFP light. Aspergillus nidulans with device BBa_K678062- Front Aspergillus nidulans with device BBa_K678062- Back Device BBa_K678063 The 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. Aspergillus nidulans with device BBa_K678063 - DIC light. Aspergillus nidulans with device BBa_K678063 - RFP light. Aspergillus nidulans with device BBa_K678063 - Front Aspergillus nidulans with device BBa_K678063 - Back Control The control strain shows no auto-fluorescence. Wild type Aspergillus nidulans - DIC light. Wild type Aspergillus nidulans - RFP light. Wild type Aspergillus nidulans - YFP light. Wild type Aspergillus nidulans - Front Wild type Aspergillus nidulans - Back