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Overview

It is known that A. niger is an excellent producer and secretor of secondary metabolites and organic acids and is therefore a target commonly used in synthetic biology which made it an interesting subject for us as an iGEM team to work with. Resulting from its common use as a secreting organism its infrastructure is attractive because its functionality is crucial for good production and secretion. This is why we labeled the cytoskeleton and the septa by fusing actin, for visualization of the actin cytoskeleton, and a H+-ATPase located in the septa with GFP. The GFP was introduced at the n-terminus and led to the possibility to observe the structures under a fluorescent microscope.

Introduction

The visualization of the actin cytoskeleton of A. niger is used as a first step towards visualization of the infrastructure. It is known that A. niger is a great producer and secretor of secondary metabolites and organic acids and its infrastructure is crucial for this processes.

The actin cytoskeleton is known to be needed for the maintenance of the shape of the cells, to adhere to substances and to help during growth and secretion. It exists of actin cables which are long, thin, parallel fibers and cortical actin patches which are highly polarized. The actin patches are also known to be present in growing tips and help in a structure called Spitzenkörper with growth and secretion. Vesicles are delivered towards the growing tips and its lipids are used for growth. The vesicles are moved towards the Spitzenkörper with the help of microtubules and the actin filaments take them over.

The septa is a permeable membrane between neighboring cells allowing transport and communication between the cells. This structure is ring like and can open and close depending on what is needed at that moment. It is build up of three structures which are actins, septins and formins. All three structures are proven to be crucial for the proper formation of the septal band and work closely together.

Aim

The goal is to visualize trafficking in A. niger by fusing the actin cytoskeleton and a H+-ATPase located in the septa with a fluorescent protein such as GFP on the n-terminus.

Approach

For the visualization of the actin cytoskeleton and the septa (by using a H+-ATPase) the genes had to be fused with GFP. To do so a first step was to isolate the DNA from A. niger as the genes are already present in the genomic DNA. Afterwards the genes were amplified using PCR. The primer pairs used for the amplification also added restriction sites to genes that were needed for the ligation into the desired vector later on.

The second part was to build the vector needed to successfully transform A. niger. At first the genes obtained via PCR were ligated into a pJET vector. This allowed us to check via sequencing that no mistakes were made during the PCR. After confirmation of the gene it was grown, and isolated from E. coli cut out of the pJET vector and ligated into a house internal brick system. The house internal brick system contains, as shown in picture 1, a GFP fusion on the n-terminus and a xlnD promoter and terminator. This promoter is induced by xylose. Furthermore the vector contains to selection markers. The ampicillin used in E. coli cloning steps and the pyr gene used for selection in A. niger which leads to an organism that no longer depends on uridine.

Last but not least after obtaining the good house internal brick it was introduced into A. niger. This mutant was grown and replated twice to obtain single colonies. To check the presence of the desired insert the genomic DNA was isolated and this was followed by a PCR using a GFP forward and an actin reverse primer and only GFP and only actin primers. After confirmation of the presence pictures could be made under a fluorescent microscope observing the structures.

Results

Septa visualization

For the visualization of the septa we got the desired construct which means we obtained the gene and showed that it is the right one and ligated into the house internal brick system with a GFP fused on the n-terminus. This is also shown in the gel picture below which displayes the results from restriction digestion showing the desired bands of ̴7100bp for the vectior and ̴3100bp for the ATPase gene. But unfortunately transformation into A. niger did not work out as planned and we did not get any further that having the construct in E. coli but not in A. niger.

Actin cytoskeleton visualization

The visualization of the actin cytoskeleton started with making a construct containing GFP and the actin encoding gene. To get the construct the gene was isolated and amplified from genomic DNA of A. niger and ligated into pJET. After seqeuencing verification of the actin gene it was ligated into a house internal brick which already contained a GFP fusd to the n-terminus, In earlier experiments this was shown. The correctness of the construct was checked using restriction digestion and the expected bands of ̴1900bp for the actin gene and ̴7100bp for the house internal brick were seen on the gel. The obtained vector was than introduced into A. niger and the transformation was checked using PCR on genomic DNA isolated from the transformants. We were able to show the presence of GFP in one of the transformants but unfortunately the primer combination of a GFP forward primer and an actin reverse primer did not work as planned and we were unable to confirm the presence of the construct in any other transformants. But two of the samples were grown for micrscopy of which one was the one where the presence of GFP was shown. And microscopy showed that only that sample contained the desired actin GFP fusion.

Conclusions

Visualization of the septa

For the visualization of the septa we have a construct containing the gene of interest and an n-terminal GFP fusion. In theory the construct is ready to be transformed into A. niger but transformation failed two times. Thus it has to be checked again before it is ready to be introduced into A. niger.

Figure 1: Restriction digestion of the ATPase gene in the vector p>

Visualization of the actin cytoskeleton

The visualization of the actin cytoskeleton worked out as planned. We managed to build a construct containing the actin encoding gene fused to a GFP on the n-terminus. This is present in a house internal brick. Furthermore transformation of A. niger worked out and single colonies were obtained. After harvesting spores it was possible to observe the structures under a fluorescent microscope and evaluate them. As expected we saw that the actin cytoskeleton is needed to move organelles through the cells and therefore wraps around the organelles. Furthermore we were able to show the presence of actin in the septa and we it was possible to see the continuation of the actin cytoskeleton throughout the cells.

Actin cytoskeleton wrapping around an organellep>