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Outline

It is known that A. niger is an excellent producer and secretor of secondary metabolites and organic acids. As a target 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 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 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. It is known that A. niger is a great producer and secretor of secondary metabolites and organic acids and its infrastructure is crucial for these 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 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 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. This would include communication in stress situations as well as shutting of the neighboring cell when something is wrong with it. It is built 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) 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.

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 two selection markers. The ampicillin resistance gene used in E. coli cloning steps and the pyrA gene are used for selection in A. niger. The pyrA gene leads to an organism that no longer depends on uridine.

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 a GFP forward primer and an actin reverse primer, only GFP or only actin primers. After confirmation of their presence pictures could be made under a fluorescent microscope observing the structures.

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 vectior and ̴3100bp for the ATPase gene. Unfortunately transformation into A. niger did not work out as planned and we did not get any further than having the construct in E. coli.

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 micrscopy 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.

Conclusions

Visualization of the septa

For the visualization of the septa we have a construct containing the proton-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

Unfortunately the construct still contains an illegal restriction site which kept us from sending it to the parts registry. The illegal restriction site is present in an intron and one cannot estimate what happens when changing it. This is a problem that arises with the use of eukaryotes and additional actions have to be taken. The primers for site directed mutagenesis arrived and it appears to be working. Therefore we will be able to send in the GFP actin construct soon.