Proof of concept

Five different plasmids were constructed with the Plug 'n' Play assembly standard in order to verify the systems function in filamentous fungi. To ensure a successful transformation in fungi the backbone plasmid pFun has two NotI restriction sites flanking the device to be inserted. Hereby, the device can be cut out of the plasmid and a linearised DNA fragment can be transformated into the fungus. The devices for proof of concept in fungi are not design to be inserted at a specific site in the fungal genome. Therefore, the device can be integrated at any site and with a random number of copies by non homologous end-joining (NHEJ) in the fungus. This means that the possibility of the disruption of essential genes exists.

The fungal proof of concept plasmids, were constructed with the same strong constitutive promoter PgpdA, the TtrpC terminator, and the pyrG marker cassette. Different genes encoding fluorescent proteins were included in the reporter system, where different compartments of fungi could be targeted. The reporter system was designed to target the nucleus, peroxisomes, and the mitochondria. All transformations were performed in the Aspergillus nidulans laboratory strain: argB2, pyrG89, veA1.

We have proved that the Plug 'n' Play assembly standard can be easily applied for the construction fungal plasmids. The constructed plasmids were transformed into A. nidulans and in each case the fluorescent protein was expressed. Only the strain transformed with the plasmid targeting GFP to the mitochondria did not result in transform ants and due to time limitation the experiment was not repeated.

The obtained transformants with the different constructed devices integrated into the genome all had a different morphology and were growing slower than the wild type strain. Especially the strain transformed with pJEJAM14 looked sick. The fungi also had a pronounced number of undefined vacuoles that can be seem on the images obtained from differential interference contrast (DIC) microscopy. Such 'symptoms' are not uncommon when DNA is introduced into the genome by NHEJ. Below the images of the microscopy are presented.

pJEJAM12 BBa_K678060

pJEJAM12 is a plasmid intended for linearization and transformation into A. nidulans. The plasmid contains the gene encoding green fluorescent protein (GFP) under the control of the strong constitutive PgpdA promoter. The expressed GFP was evenly spread in the hyphae.

DIC image of Aspergillus nidulans with pJEJAM12.	Fluorescence image of Aspergillus nidulans with pJEJAM12.

Aspergillus nidulans with pJEJAM12 - Shown from front	Aspergillus nidulans with pJEJAM12 - Shown from Back

pJEJAM13 BBa_K678061

pJEJAM13 is a plasmid intended for linearization and transformation into A. nidulans. The plasmid contains the gene encoding green fluorescent protein (GFP) with the peroxisomal targeting sequence 1 (PTS1) fused to the C-terminus, under the control of the strong constitutive PgpdA promoter.

Green fluorescence can be observed in the hyphae and in clear spots.This indicates that GFP is targeted to the peroxisomes, but further investigations are required to confirm this. The 'background' fluorescence was however not expected and a replication of the experiment should be conducted in order to be able to draw any conclusions upon this.

DIC image of Aspergillus nidulans with pJEJAM13.	Fluorescence image of Aspergillus nidulans with pJEJAM13. Green fluorescence can be observed in the hyphae and in clear spots.

Aspergillus nidulans with pJEJAM13 - Shown from front	Aspergillus nidulans with pJEJAM13 - Shown from back

pJEJAM14 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 pJEJAM14 plasmid holding device BBa_K678062, are cut at the two NotI site before transformation, ensuring linearised DNA fragment for optimal result.

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

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

pJEJAM15 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.

DIC image ofAspergillus nidulans with device BBa_K678063 - detected with DIC light.	Fluorescence image of 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

Wild type

DIC image of Wild type Aspergillus nidulans .	RFP fluorescence image of wild type Aspergillus nidulans .	GFP fluorescence image of wild type Aspergillus nidulans .

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