Latest revision as of 20:04, 21 September 2011

Proof of concept - Fungi

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 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 the linearized DNA fragments can be transformed into the fungus. The devices for proof of concept are not designed 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). This means that the possibility of the disruption of essential genes exists.

The fungal proof of concept plasmids, were constructed with the 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 here prove that the Plug 'n' Play assembly standard can easily be 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 pJEJAM16 targeting GFP to the mitochondria did not result in any transformants 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 seen 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

pJEJAM14 is a plasmid intended for linearization and transformation into A. nidulans. The plasmid contains the gene encoding monomeric red fluorescent protein (mRFP1) under the control of the strong constitutive PgpdA promoter. The expressed mRFP1 was evenly spread in the hyphae.


DIC image of Aspergillus nidulans with pJEJAM14.	Fluorescence image of Aspergillus nidulans with pJEJAM14.


Aspergillus nidulans with pJEJAM14 - shown from front	Aspergillus nidulans with pJEJAM14 - shown from back

pJEJAM15 BBa_K678063

pJEJAM15 is a plasmid intended for linearization and transformation into A. nidulans. The plasmid contains the gene encoding monomeric red fluorescent protein (mRFP1) with a nucleosomal targeting sequence (NLS) fused to the C-terminus, under the control of the strong constitutive PgpdA promoter.

Red fluorescence can be observed in clear spots. To confirm that the mRFP1 in fact is targeted to the nucleus further experiments have to be conducted.


DIC image ofAspergillus nidulans with pJEJAM15.	Fluorescence image of Aspergillus nidulans pJEJAM15.


Aspergillus nidulans with pJEJAM15 - Shown from front	Aspergillus nidulans with pJEJAM15 - Shown from back

Wild type

The wild type strain shows no background or auto-fluorescence.


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

@@ Line 64: / Line 64: @@
 <div id="menu">
 <!--<span id="test">test</span>-->
-<a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/fungi#Filamentous Fungi" class="h1">Filamentous Fungi</a><br><br>
-<a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/fungi#Growth of filamentous fungi" class="h2">Growth of filamentous fungi</a><br><br>
-<a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/fungi#Aspergillus nidulans" class="h2">Aspergillus nidulans</a><br><br>
-<a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/fungi#Gene targeting in fungi" class="h2">Gene targeting in fungi</a><br><br>
 <a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/fungi#Proof of concept" class="h1">Proof of concept</a><br><br>
-<a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/fungi#Device BBa_K678060" class="h2"> pJEJAM12 BBa_K678060</a><br><br>
+<a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/fungi#pJEJAM12 BBa_K678060" class="h2"> pJEJAM12<br>
-<a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/fungi#Device BBa_K678061" class="h2"> pJEJAM13 BBa_K678061</a><br><br>
+ BBa_K678060</a><br><br>
-<a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/fungi#Device BBa_K678062" class="h2"> pJEJAM14 BBa_K678062</a><br><br>
+<a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/fungi#pJEJAM13 BBa_K678061" class="h2"> pJEJAM13 <br>
-<a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/fungi#Device BBa_K678063" class="h2">pJEJAM15 BBa_K678063</a><br><br>
+BBa_K678061</a><br><br>
-<a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/fungi#Control strain" class="h2"> Control strain</a><br><br>
+<a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/fungi#pJEJAM14 BBa_K678062" class="h2"> pJEJAM14 <br>
-<a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/fungi#References" class="h1"> References</a><br>
+BBa_K678062</a><br><br>
+<a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/fungi#pJEJAM15 BBa_K678063" class="h2"> pJEJAM15 <br>
+BBa_K678063</a><br><br>
+<a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/fungi#Wild type" class="h2"> Wild type</a><br><br>
@@ Line 84: / Line 84: @@
-<br>
+<a name="Proof of concept"></a><h1><b>Proof of concept</b></h1>
-<a name="Filamentous Fungi"></a><h1><b>Filamentous Fungi</b></h1>
 <p align="justify">
-Fungi 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. <br>
+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 the backbone plasmid <a href="http://partsregistry.org/Part:BBa_K678046">pFun</a> has two NotI restriction sites flanking the device to be inserted. Hereby, the device can be cut out of the plasmid and the linearized DNA fragments can be transformed into the fungus. The devices for proof of concept are not designed 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). This means that the possibility of the disruption of essential genes exists.<br><br>
-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. </p><br>
+The fungal proof of concept plasmids, were constructed with the strong constitutive promoter <a href="http://partsregistry.org/Part:BBa_K678024">P<i>gpdA</i></a>, the <a href="http://partsregistry.org/Part:BBa_K678036">T<i>trpC</i></a> terminator, and the <a href="http://partsregistry.org/Part:BBa_K678040"><i>pyrG marker cassette</i></a>. 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 <i> Aspergillus nidulans</i> laboratory strain:<i> argB2, pyrG89, veA1</i>. </p>
-<a name="Growth of filamentous fungi"></a><h3><b>Growth of filamentous fungi</b></h3>
-<p align="justify">
-The vegetative growth of filamentous fungi starts with the germination of a spore when the conditions are right . The spore germination leads to formation of hyphae (1). A fungal hypha is a long tubular modular structure composed of individual cells (2). Hyphae extend only at their tips and are typically divided into individual cellular compartments by the formation of septa (3). </p>
-<p align="center">
-<img src=" https://static.igem.org/mediawiki/2011/7/7a/Germlig-hyphae.png" height="110px" > </img>
-<p align="justify">
-Filamentous fungi grow by the polar extension of hyphae and multiply by branching (4). The branched hyphae forms a network of interconnected cells called a mycelium (1). The mycelium forms a radially symmetric colony that expands over large area until growth is limited by for example lack of nutrients (1,5). The fungal mycelium appears to be a formless collection of corresponding vegetative cells. However, the various cells within the mycelium interact to form an ordered network with different hyphae or cells playing distinct roles in the acquirement of nutrients from the environment and development (1). </p>
 <br>
-<a name="Aspergillus nidulans"></a><h3><b><i>Aspergillus nidulans</i></b></h3>
 <p align="justify">
-The filamentous fungus <i>Aspergillus nidulans</i> is a widely recognised model organism (3). <i>A. nidulans</i> possesses today, in contrast to most other aspergilla, a well characterized sexual cycle and a well-developed genetics system (6). Furthermore, in <i>A.nidulans</i> the parasexual cycle has been extensively utilized. Parasexual genetic involves examination of recombination in the absence of sexual reproduction.<br>
+We here prove that the Plug 'n' Play assembly standard can easily be applied for the construction fungal plasmids. The constructed plasmids were transformed into <i>A. nidulans</i> and in each case the fluorescent protein was expressed. Only the strain transformed with <a href="http://partsregistry.org/Part:BBa_K678064">pJEJAM16</a> targeting GFP to the mitochondria did not result in any transformants and due to time limitation the experiment was not repeated.<br><br>
-<br>
-The genetic analysis has produced a deep understanding of both the physiology of Aspergillus and the organisation of the genome (7). This research has advanced the study of eukaryotic cellular physiology and contributed to our understanding of metabolic regulation, development, DNA repair, morphogenesis, and human genetics diseases (6). Furthermore, the recent sequencing of the complete genome of <i>A.nidulans </i>has created a tremendous potential to obtain insight into important aspects of fungal biology such as transcriptional regulation, secondary metabolite production and pathogenicity (8). </p>
+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 seen 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.
-<br><br>
-<a name="Gene targeting"></a><h2><b>Gene targeting</b></h2>
-<p align="justify">
-Accurate manipulation of genes is a key approach in fungal molecular biology (9). The method of gene targeting facilitates precise genome manipulations which are called site directed alterations. They are performed by use of deletions, replacements and insertion of the target locus. Gene targeting is achieved by transforming fungi with a suitable linear DNA fragment that contains sequences that are identical to the target site in the genome, see figure. Different DNA fragment are constructed depending on deletion, replacement or insertion. The fungi can integrates linear DNA fragment in its genome by using its repair system of DNA double stranded breaks. </p>
-<br>
-<p align="center">
-<img src="https://static.igem.org/mediawiki/2011/c/c1/Figure_8.JPG" height="110px" > </img>
-</p>
-<br>
-<p align="justify">
-Two mechanisms of DNA double strand break repair ensure that the introduced piece of DNA is pasted into the fungal genome, to be replicated stably: Homologous Recombination and Non Homologous End Joining, also called illegitimate recombination (9). Homologous recombination involves interaction between homologous sequences, whereas NHEJ involves ligation of the strand ends independently of DNA homology (10). Precise genome manipulation can often be tedious and time-consuming, because fungi appear to favour Non Homologous End Joining over Homologous recombination resulting in low gene targeting efficiencies (9). </p>
-<br><br>
-<a name="Proof of concept"></a><h1><b>Proof of concept</b></h1>
-<p align="justify">
-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 <a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a> 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.<br><br>
-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 <i> Aspergillus nidulans</i> stain:<i> argB2, pyrG89, veA1</i> by random non-homologues-end-joining integration, since we only want to verify the reporter gene by fluorescence microscopy. </p>
-<br>
-<br>
-<p align="justify">
-We succeeded in proving that the Plug 'n' Play assembly system is easily applied for constructing fungal vectors. Transformation and expression in fungi succeed by expressing the two fluorescence proteins GFP and RFP in <i>A. nidulans </i> not localized to any compartment. Furthermore, expression of fluorescence proteins GFP and RFP in <i>A. nidulans </i>localized to the nucleus and the peroxiomes succeeded. However, the transformation of the fungi with the mitochondrial targeting signal did not succeed.
-<br><br>
-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, therefore the fungi also showed a pronounced number of unspecific vacuoles. The change occur due to the DNA fragment were randomly integrated and have inflicted some pathways in the fungi. However, this was to expect when using integration by random non-homologues-end-joining.
 </p>
 <br>
 <br>
-The results of the created reporter system for fungi are displayed below.
 <br>
-<br>
+<a name="pJEJAM12 BBa_K678060"></a><h2>pJEJAM12 BBa_K678060</h2>
-<a name="Device BBa_K678060"></a><h2>Device BBa_K678060</h2>
 <p align="justify">
-Observed is green fluorescence spread evenly in the hyphae, which correlate with what expected for the device <a href="http://partsregistry.org/Part:BBa_K678060">BBa_K678060</a> that holds the gene for green fluorescence protein GFP with no specific targeting signal.
+<a href="http://partsregistry.org/Part:BBa_K678060">pJEJAM12</a> is a plasmid intended for linearization and transformation into <i>A. nidulans</i>. The plasmid contains the gene encoding green fluorescent protein (GFP) under the control of the strong constitutive P<i>gpdA</i> promoter. The expressed GFP was evenly spread in the hyphae.
 </p>
 <br>
-<br>
 <p align="center">
-<img src="https://static.igem.org/mediawiki/2011/2/20/Device_22.png" height="100px" align="center"> </img>
+<img src="https://static.igem.org/mediawiki/2011/e/ef/Device_22_ny.png" height="100px" align="center"> </img>
 <br>
@@ Line 164: / Line 120: @@
 </tr>
 <tr>
-<td><i>Aspergillus nidulans </i> with device <br><a href="http://partsregistry.org/Part:BBa_K678060">BBa_K678060</a> - detected with DIC light.</td>
+<td>DIC image of<i> Aspergillus nidulans </i> with <br><a href="http://partsregistry.org/Part:BBa_K678060">pJEJAM12</a>.</td>
-<td><i>Aspergillus nidulans </i> with device <br><a href="http://partsregistry.org/Part:BBa_K678060">BBa_K678060</a> - detected with GFP filter.</td>
+<td>Fluorescence image of <i>Aspergillus nidulans </i> with <br><a href="http://partsregistry.org/Part:BBa_K678060">pJEJAM12</a>.</td>
 </tr>
 </table>
@@ Line 178: / Line 134: @@
 </tr>
 <tr>
-<td><i>Aspergillus nidulans </i> with device <br><a href="http://partsregistry.org/Part:BBa_K678060">BBa_K678060</a> - Shown from front</td>
+<td><i>Aspergillus nidulans </i> with <br><a href="http://partsregistry.org/Part:BBa_K678060">pJEJAM12</a> - Shown from front</td>
-<td><i>Aspergillus nidulans </i> with device <br><a href="http://partsregistry.org/Part:BBa_K678060">BBa_K678060</a> - Shown from Back </td>
+<td><i>Aspergillus nidulans </i> with <br><a href="http://partsregistry.org/Part:BBa_K678060">pJEJAM12</a> - Shown from Back </td>
 </tr>
 </table>
@@ Line 188: / Line 144: @@
 <br>
-<a name="Device BBa_K678061"></a><h2>pJEJAM13 BBa_K678061</h2>
+<a name="pJEJAM13 BBa_K678061"></a><h2>pJEJAM13 BBa_K678061</h2>
 <p align="justify">
-Observed is green fluorescence spread in the hyphae and in clear spots. The occurrence of clear spots and compared to the results from device <a href="http://partsregistry.org/Part:BBa_K678060">BBa_K678060</a>, correlate with what expected for the device <a href="http://partsregistry.org/Part:BBa_K678061">BBa_K678061</a> that holds the gene for green fluorescence protein GFP with the targeting signal for the peroxiomes. 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 <a href="http://partsregistry.org/Part:BBa_K678060">BBa_K678060</a>. </p>
+<a href="http://partsregistry.org/Part:BBa_K678061">pJEJAM13</a> is a plasmid intended for linearization and transformation into <i>A. nidulans</i>. 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 P<i>gpdA</i> promoter.</p>
-<br>
 <br>
 <p align="center">
-<img src="https://static.igem.org/mediawiki/2011/9/99/Device_23.png" height="100px" align="center"> </img>
+<img src="https://static.igem.org/mediawiki/2011/6/68/Device_23_ny.png" height="100px" align="center"> </img>
+<br>
+<br>
+<p align="justify">
+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. </p>
 <br>
 <br>
@@ Line 206: / Line 166: @@
 </tr>
 <tr>
-<td><i>Aspergillus nidulans </i> with device <br><a href="http://partsregistry.org/Part:BBa_K678061">BBa_K678061</a> - detected with DIC light.</td>
+<td>DIC image of <i>Aspergillus nidulans </i> with <br><a href="http://partsregistry.org/Part:BBa_K678061">pJEJAM13</a>.</td>
-<td><i>Aspergillus nidulans </i> with device <br><a href="http://partsregistry.org/Part:BBa_K678061">BBa_K678061</a> - detected with GFP filter.</td>
+<td>Fluorescence image of <i>Aspergillus nidulans </i> with <br><a href="http://partsregistry.org/Part:BBa_K678061">pJEJAM13</a>. Green fluorescence can be observed in the hyphae and in clear spots.</td>
 </tr>
 </table>
@@ Line 218: / Line 178: @@
 </tr>
 <tr>
-<td><i>Aspergillus nidulans </i> with device <br><a href="http://partsregistry.org/Part:BBa_K678061">BBa_K678061</a> - Shown from front</td>
+<td><i>Aspergillus nidulans </i> with <br><a href="http://partsregistry.org/Part:BBa_K678061">pJEJAM13</a> - Shown from front</td>
-<td><i>Aspergillus nidulans </i> with device <br><a href="http://partsregistry.org/Part:BBa_K678061">BBa_K678061</a> - Shown from back </td>
+<td><i>Aspergillus nidulans </i> with <br><a href="http://partsregistry.org/Part:BBa_K678061">pJEJAM13</a> - Shown from back </td>
 </tr>
 </table>
@@ Line 228: / Line 188: @@
-<a name="Device BBa_K678062"></a><h2>pJEJAM14 BBa_K678062</h2>
+<a name="pJEJAM14 BBa_K678062"></a><h2>pJEJAM14 BBa_K678062</h2>
-The results shows fluorescence spread evenly in the hyphae which correlate with that in device <a href="http://partsregistry.org/Part:BBa_K678061">BBa_K678061</a>, the gene of interest, only consists of a green fluorescence protein RFP with no specific target.
+<p align="justify">
-<br>
+<a href="http://partsregistry.org/Part:BBa_K678062">pJEJAM14</a> is a plasmid intended for linearization and transformation into <i>A. nidulans</i>. The plasmid contains the gene encoding monomeric red fluorescent protein (mRFP1) under the control of the strong constitutive P<i>gpdA</i> promoter. The expressed mRFP1 was evenly spread in the hyphae.
+</p>
 <br>
 <p align="center">
-<img src="https://static.igem.org/mediawiki/2011/8/8b/Device_24.png" height="100px" align="center"> </img>
+<img src="https://static.igem.org/mediawiki/2011/a/aa/Device_24_ny.png" height="100px" align="center"> </img>
 <br>
@@ Line 246: / Line 208: @@
 </tr>
 <tr>
-<td><i>Aspergillus nidulans </i> with device <br><a href="http://partsregistry.org/Part:BBa_K678062">BBa_K678062</a> - detected with DIC light.</td>
+<td>DIC image of <i>Aspergillus nidulans </i> with <br><a href="http://partsregistry.org/Part:BBa_K678062">pJEJAM14</a>.</td>
-<td><i>Aspergillus nidulans </i> with device <br><a href="http://partsregistry.org/Part:BBa_K678062">BBa_K678062</a>  - detected with RFP filter.</td>
+<td>Fluorescence image of <i>Aspergillus nidulans </i> with <br><a href="http://partsregistry.org/Part:BBa_K678062">pJEJAM14</a>.</td>
 </tr>
 </table>
@@ Line 258: / Line 220: @@
 </tr>
 <tr>
-<td><i>Aspergillus nidulans </i> with device <br><a href="http://partsregistry.org/Part:BBa_K678062"BBa_K678062</a> - shown from front</td>
+<td><i>Aspergillus nidulans </i> with <br><a href="http://partsregistry.org/Part:BBa_K678062">pJEJAM14</a> - shown from front</td>
-<td><i>Aspergillus nidulans </i> with device <br><a href="http://partsregistry.org/Part:BBa_K678062"BBa_K678062</a> - shown from back </td>
+<td><i>Aspergillus nidulans </i> with <br><a href="http://partsregistry.org/Part:BBa_K678062">pJEJAM14</a> - shown from back </td>
 </tr>
 </table>
@@ Line 269: / Line 231: @@
-<a name="Device BBa_K678063"></a><h2>pJEJAM15 BBa_K678063</h2>
+<a name="pJEJAM15 BBa_K678063"></a><h2>pJEJAM15 BBa_K678063</h2>
+<p align="justify">
+<a href="http://partsregistry.org/Part:BBa_K678063">pJEJAM15</a> is a plasmid intended for linearization and transformation into <i>A. nidulans</i>. The plasmid contains the gene encoding monomeric red fluorescent protein (mRFP1) with a nucleosomal targeting sequence (NLS) fused to the C-terminus, under the control of the strong constitutive P<i>gpdA</i> promoter.</p> Red fluorescence can be observed in clear spots. To confirm that the mRFP1 in fact is targeted to the nucleus further experiments have to be conducted. </p>
-The results show spots of fluorescence as in Device <a href="http://partsregistry.org/Part:BBa_K678061">BBa_K678061</a> . Device <a href="http://partsregistry.org/Part:BBa_K678063">BBa_K678063</a>  consists of RFP with a target signal to the nucleus which can explain the spots compared to the results from device <a href="http://partsregistry.org/Part:BBa_K678060">BBa_K678060</a> 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.
-<br>
 <br>
 <p align="center">
-<img src="https://static.igem.org/mediawiki/2011/a/ac/Device_25.png" height="100px" align="center"> </img>
+<img src="https://static.igem.org/mediawiki/2011/e/e4/Device_25_ny.png" height="100px" align="center"> </img>
 <br>
@@ Line 287: / Line 250: @@
 </tr>
 <tr>
-<td><i>Aspergillus nidulans </i> with device <br><a href="http://partsregistry.org/Part:BBa_K678063">BBa_K678063</a> - detected with DIC light.</td>
+<td>DIC image of<i>Aspergillus nidulans </i> with <br><a href="http://partsregistry.org/Part:BBa_K678063">pJEJAM15</a>.</td>
-<td><i>Aspergillus nidulans </i> with device <br><a href="http://partsregistry.org/Part:BBa_K678063">BBa_K678063</a> - detected with RFP filter.</td>
+<td>Fluorescence image of <i>Aspergillus nidulans </i><br><a href="http://partsregistry.org/Part:BBa_K678063">pJEJAM15</a>.</td>
 </tr>
 </table>
@@ Line 299: / Line 262: @@
 </tr>
 <tr>
-<td><i>Aspergillus nidulans </i> with device <br> <a href="http://partsregistry.org/Part:BBa_K678063">BBa_K678063</a> - Shown from front</td>
+<td><i>Aspergillus nidulans </i> with <br> <a href="http://partsregistry.org/Part:BBa_K678063">pJEJAM15</a> - Shown from front</td>
-<td><i>Aspergillus nidulans </i> with device <br> <a href="http://partsregistry.org/Part:BBa_K678063">BBa_K678063</a> - Shown from back </td>
+<td><i>Aspergillus nidulans </i> with <br> <a href="http://partsregistry.org/Part:BBa_K678063">pJEJAM15</a> - Shown from back </td>
 </tr>
 </table>
@@ Line 308: / Line 271: @@
 <br>
-<a name="Control"></a><h2>Control strain</h2>
+<a name="Wild type"></a><h2>Wild type</h2>
-The control strain shows no auto-fluorescence.
+The wild type strain shows no background or auto-fluorescence.
 <p align="center">
 <table cellpadding="10px" align="center">
@@ Line 320: / Line 283: @@
 </tr>
 <tr>
-<td> Wild type <i>Aspergillus nidulans </i> - detected with DIC light. </td>
+<td> DIC image of wild type <i>Aspergillus nidulans </i>. <br></td>
-<td>Wild type <i>Aspergillus nidulans </i> - detected with RFP filter. </td>
+<td>RFP fluorescence image of wild type <i>Aspergillus nidulans </i>. <br> </td>
-<td>Wild type <i>Aspergillus nidulans </i> - detected with GFP filter.</td>
+<td>GFP fluorescence image of wild type <i>Aspergillus nidulans </i>.<br></td>
 </tr>
 </table>
@@ Line 333: / Line 296: @@
 </tr>
 <tr>
-<td>Wild type <i>Aspergillus nidulans </i> - Shown from front</td>
+<td>Wild type <i>Aspergillus nidulans </i> <br>- Shown from front</td>
-<td>Wild type <i>Aspergillus nidulans </i> - Shown from back </td>
+<td>Wild type <i>Aspergillus nidulans </i> <br>- Shown from back </td>
 </tr>
 </table>
-<br><br>
-<a name="References"></a><h1><b>References</b></h1>
-<p align="justify">
-[1]  Adams, T.H., Wieser, J.K. & Yu, J.-H., 1998. Asexual Sporulation in Aspergilus nidulans. Microbiology and Molecular Biology Reveiws, vol. 62, no. 1, pp.35-54.
-<br><br>
-[2] Harris, S.D., 1997. The Duplication Cycle in Asoergillus nidulans. Fungal Genetics and Biology, vol. 22, no. 1, pp.1-12.
-<br><br>
-[3] Harris, S.D. et al., 2009. Morphology and development in Aspergillus nidulans: A complex puzzle. Fungal Genetics and Biology, vol. 46, no. 1, sup. 1, pp.S82-92.
-<br><br>
-[4] Timberlake, W.E., 1990. Molecular Genetics of Aspergillus Development. Annual Review of Genetics, vol. 24, pp.5-36.
-<br><br>
-[5] Nielsen, J.B., 2008. Understanding DNA repair in Aspergillus nidulans - paving the way for efficient gene targeting. Technical University of Denmark.
-<br><br>
-[6] Galagan, J.E. et al., 2005. Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae. Nature, vol. 438, no. 7971, pp.1105-15.
-<br><br>
-[7] Doonan, J.H., 1992. Cell division in Aspergillus. Journal of Cell Science, vol. 103, no. 3, pp.599-611.
-<br><br>
-[8] Nielsen, M.L. et al., 2006. Efficient PCR-based gene targeting with a recyclable marker for Aspergillus nidulans. Fungal Genetics and Biology, vol. 43, no. 1, pp.54-64.
-<br><br>
-[9] Krappmann, S., 2007. Gene targeting in filamentous fungi: the benefits of impaired repair. Fungal Biology Reviews, vol. 21, no. 1, pp.25-29.
-<br><br>
-[10] Ninomiya, Y., Suzuki, K., Ishii, C. & Inoue, H., 2004. Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining. PNAS, vol. 101, no. 33, pp.12248-53.
-<br><br>
-</p>
 </td>