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Outline

A. niger is an excellent producer and secretor of secondary metabolites and organic acids. As an organism commonly used in synthetic biology it represents an interesting host for us as an iGEM team to work with. Resulting from its common use as a secreting organism its infrastructure is attractive. This is because the functionality of the infrastructure 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 allowed us the observation of 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. The actin cytoskeleton is needed for the maintenance of the shape of the cells, to adhere to substances and to help during growth and secretion. It consists 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 septum 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. This includes communication in stress situations as well as shutting off the neighboring cell when something is wrong with it. The septum is built up of three structures, which are actins, septins and formins. All three structures have been proven to be crucial for the proper formation of the septal band and work closely together[1].

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.p>

Approach

For the visualization of the actin cytoskeleton and the septa (by using a H+-ATPase) those 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. This vector contains a pyrA gene which is an uridine-auxotrophy marker which means that after introduction of this vector into A. niger it is no longer dependent on uridine. This is used to check easily whether transformation worked.

The second part was to build the vector needed to successfully transform AA. 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 E. coli containing the plasmid was grown, and the plasmid was isolated from E. coli. Then the desired gene was cut out of the pJET vector and ligated into an in-house brick system. The in-house 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 two selection markers. The ampicillin resistance gene used in E. coli cloning steps and the pyrA gene used for selection in A. niger.

Last but not least, after obtaining the in-house brick it was introduced into A. niger. This mutant was grown and re-plated twice to obtain pure single colonies. To check the presence of the desired insert the genomic DNA was isolated and this was followed by a PCR using three sets of primers, (i) a GFP forward primer and an actin reverse primer, (ii) only GFP primers, or (iii) only actin primers. After confirmation of the presence of the inserts (GFP and actin), pictures were made using a fluorescent microscope.

Schematic representation of the construct used to transform A. niger.

Results

Septa visualization

For the visualization of the septa we got the desired construct. This means that we obtained the proton-ATPase encoding gene and showed that it is the right one by sending it to sequencing. Afterwards it was ligated into the in-house brick system with a GFP fused on the N-terminus. This is also shown in the gel picture below which displays the results from restriction digestion showing the desired bands of ̴7100bp for the vector and ̴3100bp for the ATPase gene. Unfortunately, we were unable to successfully transform A. niger with the construct containing the ATPase gene.

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 the cloning vector pJET. After sequence verification of the actin gene it was ligated into an in-house brick which already contained a GFP fused to the N-terminus. In earlier experiments of the technician Tom Schonewille of the lab 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 microscopy imaging, of which one was the sample containing the GFP as was shown by PCR earlier. Microscopy showed that only that sample contained the desired actin GFP fusion.

The video displays the depth of the mycelium with a septa in between.

The video shows the depth of a bigger part of the mycelium.

Discussion

Septa visualization

For the construct containing the H+-ATPase gene quite some effort was done. With around 3000bp the gene is of good length. This led to some complications in the construction of the vector. Ligation times had to be increased and the chance of mistakes in the PCR was higher. But all on all everything went smoothly. At least until the transformation of A. niger. At that point no progress was made. The construct is available but not yet introduced into A. niger. Transformation was tried twice but without success.

Actin cytoskeleton visualization

The actin cytoskeleton is made visible by fusing it to GFP. A construct was designed containing an N-terminal GFP fusion to the actin. Obtaining the actin and building the construct took place with some obstacles. The biggest problem was to amplify the gene because it appeared later on that the primers were incorrect. But after this was noticed the amplification was no longer a problem. Afterwards the project went on smoothly and no big difficulties were met. In addition the transformation of A. niger with the construct was not a problem. But unfortunately the confirmation that the construct was present in the transformants was not as easy as we thought at first glance. It looked like the primer combination used was not working. For one sample the presence of GFP was confirmed and this and another sample were grown for microscopy. Luckily the sample contained the desired construct and pictures were obtained.

Conclusions

Visualization of the septa

For the visualization of the septa we have a construct containing the H+-ATPase gene 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 an in-house brick. Furthermore transformation of A. niger worked out and single colonies were obtained. After harvesting spores it was possible to observe the structures of the actin cytoskeleton 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 observe the continuation of the actin cytoskeleton throughout the cells.

Actin cytoskeleton wrapping around an organelle

References

1.Harris, Steven D. (2001). Septum formation in Aspergillus nidulans Current opinion in Microbiology, 4(6), 736-739.