Team:Wageningen UR/Notebook

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            <h3>work in progress</h3>

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             <p>The restriction enzymes that we ordered last week have arrived. After a few attempts the sequences have been optimized and the G-blocks can be ordered. Also some Lovastatin has been ordered to perform some experiments with A. niger. Lovastatin may be fatal to cell growth of A. niger therefore experiments have to indicate whether A. niger is resistant to Lovastatin. Otherwise A. niger has to be transformed with the resistance gene from A. terreus.</p>

             <p> In order to expand the database, more information on secondary metabolite backbone enzymes from Aspergilli needs to be added. Luckily there is a recent publication on secondary metabolites from A. nidulans, A. fumigatus, A. niger and A. oryzae. <br /> <br />

- D.O. Inglis et al., 2013. <i> Comprehensive annotation of secondary metabolite biosynthetic genes and gene clusters of Aspergillus nidulans, A. fumigatus, A. niger and A. oryzae. </i> BMC Microbiology, Vol. 13, p. 1-23.

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             <p class="date">1.08.2013</p>

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             <p> This week we ordered the restriction enzymes for the assembly strategy. Also other stuff should be ordered like pJET, lovastatin, A. niger & A. terrus strain, Gibson assembly kit...

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             <h3>Obtained E.coli expressing chromoproteins from Braunschweig</h3>

             <p>We obtained the E.coli from Braunschweig that have plasmids with an chromoprotein encoding gene insert. Lets start introducing it into Aspergillus Niger!</p>

             <p> In a similar way that the DAPI stained cells give an indication of metabolic activity by showing an increase in nuclei, GFP-transformed A. niger also give an indication of metabolic activity by giving an indication on whether transcription and translation occur.

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             <p> text here

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             <p>Finally the search has begun, this week we have started with literature research for the Lovastatin project. Some topics were for example what is Lovastatin, which genes are involved in Lovastatin production in Aspergillus terreus, and how to measure Lovastatin production. The first idea was to introduce the Lovastatin pathway of A. terreus into A.niger. During the research we found that one of the bigger genes involved is composed of several domains. Our new idea now is to synthesize these domains, making several modules that we can assemble as we wish. </p>

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             <p> text here <br /> <br />

- D.O. Inglis et al., 2013 <i> Comprehensive annotation of secondary metabolite biosynthetic genes and gene clusters of Aspergillus nidulans, A. fumigatus, A. niger and A. oryzae.</i> BMC Microbiology Vol. 13 p. 1-23 <br />

- J.F. Sanchez et al., 2012. <i> Advances in Aspergillus secondary metabolite research in the post-genomic era. </i> Nat. Prod. Rep. Vol. 29, p. 351-371

             <p>After some waiting time the primers needed for the visualization of the septa using a proton-ATPase finally arrived and this part of the project could also get started by isolating the gene from genomic A. niger DNA.</p>

             <p>The actin gene for cytoskeleton visualization purposes was ligated into a house internal brick system with a n-terminal GFP fusion. This construct was then introduced into E. coli for amplification purposes.</p>

             <p>The lovastatin pathway has been added to the metabolic model of Aspergillus niger. Because the medium composition of the model is not yet properly defined, the maximum flux towards lovastatin can be achieved. This however is not realistic and therefore the next thing is to redefined the medium composition. <br /> <br />  

             <p> text here

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             <p> As described in literature, N593 formed giant cells after 24h at 44C. Since transcriptome data on N400 from multiple stages in its life cycle is available, this strain is chosen such that this research complements the current RNA landscape profile of A. niger and allows for comparison of data from different life stages. However, unlike N400, N593 repeatedly formed mycelium at 44C. To overcome this effect the temperature was increased to 45C, at which a single cell phenotype was obtained for N593.

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             <p> Different environmental conditions induce changes in phenotypic cellularity of Aspergillus niger. Mapping reads onto the reference genome allows for discovery of patterns in gene expression that are unique to the single cell phenotype.

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Finding sets of candidate genes causative to the single cell phenotype in Aspergillus niger by transcriptome analysis. <br /> <br />

- is A. niger metabolically active at 44C? <br />

- does A. niger divide at 44C?

         <img src="http://www.urmc.rochester.edu/labs/Munger-Lab/images/Kegg-Pathway.png"/>

             <p> The current metabolic model of A. niger needs to be expanded to include the lovastatin pathway as known from literature. First the model has to be checked: is it balanced? can we perform a FBA on the current model? <br /> <br />

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             <p>We reserved a room for all future Monday and Thursday lunch meetings in the Forum</p>

             <p>Finally the labwork for the visualization of the septa and actin cytoskeleton started. And the first thing to do was order primers and isolate genomic DNA from A. niger.</p>

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             <p>We created a Gmail account. Let the spam begin</p>

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             <p>We met with the iGem Uppsala 2013 team in Uppsala</p>

             <p>All the <i3>E. coli</i3> stabs with constructs from Addgene and the Imamura lab were revived and glycerol stocks were made and preserved at -80'C.</p>

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             <p>We created a Twitter account. Hello world! Meet iGem Wageningen 2013</p>

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             <p>Looked into sequencing options: codon optimization to Aspergillus Niger, suffix/prefix, (illegal) restrictionsites. Let’s order some g-blocks!</p>

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             <p>We had a lecture on good lab practice and safety in the lab</p>

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             <p>A paper from 1970 shows that at 44C Aspergillus niger does not germinate, but rather forms giant cells. This dimorphism is not unfamiliar within the fungal world, where at lower temperatures the mycelium is formed and at higher temperatures the yeast-like form is found. <br /> <br />

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