Proof of concept

Several separate plasmid was constructed by use of the Plug 'n' Play assembly system to verify the systems function in filamentous fungi. To perform a successful transformation in fungi the backbone plasmid for the fungal devices BBa_K678046 have two NotI restriction sites flanking the device. Hereby, the device can be cut and a linearised DNA fragment can be transformated into the fungi. The devices for proof of concept in fungi are not design to be inserted at a specific site at the fungal genome. Therefore, a fungal strain with intact pathway for non homologous recombination was used. Therefore, the device can integrate at any site in the fungi and thereby also at site of essential genes.

All the fungal devices for proof of concept, were constructed with the same strong constitutive promoter PgpdA, the TtrpC terminator, and the marker cassette of pyrG. Different fluorescence reporter genes was used for creating a reporter system, were different compartments of the fungi could be targeted. The compartment attempted to target was the nucleus, peroxisomes, and the mitochondria. All transformation was performed in the laboratory Aspergillus nidulans stain: argB2, pyrG89, veA1 by random Non Homologues End Joining integration, since verification of the reporter gene is only made by fluorescence microscopy.

We succeeded in proving that the Plug 'n' Play assembly system is easily applied for constructing fungal vectors. Transformation and expression of the fluorescence proteins GFP and RFP was successfully conducted in A. nidulans, not localized to any compartment.Furthermore, expression of fluorescence proteins GFP and RFP in A. nidulans localized to the nucleus and the peroxiomes succeeded. However, the transformation of the fungi with the mitochondrial targeting signal did not succeed.

The obtained transformants with integration of the four different devices changed pronounced morphology compared with the wild type control strain. The strains was visible sick and had slow growth. The fungi also showed a pronounced number of unspecific vacuoles. The changes occur due to the DNA fragments were randomly integrated and have inflicted some pathways in the fungi. However, this was not unexpect when using integration by random Non Homologues End Joining.

Device BBa_K678060

Green fluorescence can be observed evenly spread in the hyphae. This correlate with what was expected for the device BBa_K678060 that holds the gene GFP for green fluorescence and with no specific targeting signal.

The constructed pJEJAM 12 plasmid consist of device BBa_K678060 (pgdA,GFP,TrpC,pyrG) , which are cut at the two NotI site before transformation, ensuring linearised DNA fragment for optimal result.

Aspergillus nidulans with device BBa_K678060 - detected with DIC light.	Aspergillus nidulans with device BBa_K678060 - detected with GFP filter.

Aspergillus nidulans with device BBa_K678060 - Shown from front	Aspergillus nidulans with device BBa_K678060 - Shown from Back

Device BBa_K678061

Green fluorescence is can be observed in the hyphae and in clear spots. The occurrence of clear spots was expected for device BBa_K678061, which holds the gene GFP encoding green fluorescence proteins and targeting signal for the peroxiomes. This correlates fine with comparison of device BBa_K678060, which have non-targeting signal.
However, we cannot conclude that the signal is accumulated in the peroxisomes, since they are not dyed. Though, it can be concluded that the GFP signal is targeting to a specific place and accumulated somewhere in the fungi compared to the results from device BBa_K678060.

The plasmid constructed pJEJAM 13 holding device BBa_K678061, are cut at the two NotI site before transformation, ensuring linearised DNA fragment for optimal result.

Aspergillus nidulans with device BBa_K678061 - detected with DIC light.	Aspergillus nidulans with device BBa_K678061 - detected with GFP filter.

Aspergillus nidulans with device BBa_K678061 - Shown from front	Aspergillus nidulans with device BBa_K678061 - Shown from back

Device BBa_K678062

Observed is red fluorescence spread evenly in the hyphae, which correlate with what expected for the device BBa_K678062, that holds the gene for red fluorescence protein RFP with no specific targeting signal.

The constructed pJEJAM 14 plasmid holding device BBa_K678062, are cut at the two NotI site before transformation, ensuring linearised DNA fragment for optimal result.

Aspergillus nidulans with device BBa_K678062 - detected with DIC light.	Aspergillus nidulans with device BBa_K678062 - detected with RFP filter.

Aspergillus nidulans with device - shown from front	Aspergillus nidulans with device - shown from back

Device BBa_K678063

Red fluorescence can be observed in clear spots. The occurrence of clear spots and compared to the results from device BBa_K678062, correlate with what expected for the device BBa_K678063 that holds the gene for red fluorescence protein RFP with the targeting signal for the nucleus. However, we cannot conclude that the signal is accumulated in the nucleus, since they are not dyed. Though, it can be concluded that the RFP signal is targeting to a specific place and accumulated somewhere in the fungi compared to the results from device BBa_K678062.

The constructed pJEJAM15 plasmid, holding device BBa_K678063, are cut at the two NotI site before transformation, ensuring linearised DNA fragment for optimal result.

Aspergillus nidulans with device BBa_K678063 - detected with DIC light.	Aspergillus nidulans with device BBa_K678063 - detected with RFP filter.

Aspergillus nidulans with device BBa_K678063 - Shown from front	Aspergillus nidulans with device BBa_K678063 - Shown from back

Control strain

Wild type Aspergillus nidulans - detected with DIC light.	Wild type Aspergillus nidulans - detected with RFP filter.	Wild type Aspergillus nidulans - detected with GFP filter.

Wild type Aspergillus nidulans - Shown from front	Wild type Aspergillus nidulans - Shown from back