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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 and the septa of A. niger is used as a first step towards visualization of trafficking in A. niger. This is of great interest because of the function of A. niger in production of secondary metabolites and organic acids. A. niger is a great secretor and visualizing this mechanisms can help to understand the mechanisms underlying the process of production and secretion. 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 septum is a membrane that separates two neighbouring cell in a filamentous fungus such as A. niger. It is a contractile ring that can be open or closed and it allows nutrients and organelles to pass through as needed. The septal band consists of actins, septins and formins. For septation, actins are needed as they form a ring structure that is a first step in septation. Also they are needed for contraction of the ring and are associated with microtubules to do so! The septins are a conserved family of proteins and they also form a ring like structure covering the already mentioned actin ring. Furthermore they appear to be of importance for mitosis and the mitotic spindle and the mitotic signals seem to be important for the assembly of a septal band. The third component of the septa appears to be formins which are needed for the actin ring to assemble after mitosis. All in all it appears that all three components work together really closely and septation is not working properly if one of them is not functioning properly.

Aim

The aim is to visualize the actin cytoskeleton and the septa by labeling it with a fluorescent protein such as GFP.

Approach

In general the same was done for the visualization of the actin cytoskeleton and the septa of A. niger. The genes are already present in A. niger and therefore the genomic DNA was isolated from A. niger and the gene were amplified using PCR. Afterwards a construct was built with an n-terminal GFP fusion using a house internal brick system. To build this construct first the genes were ligated into pJET and verification via sequencing took place. The verified genes that were declared as good were than used to ligate them in the house internal brick system which already contained a GFP fused to the n-terminus. This brick system which also contains a selection marker for A. niger was than introduced into A. niger and via fluorescent microscopy the structures could be observed.

For the colorization of the septa a H+-ATPase is used which would be PMA1. This is done because the H+-ATPase is located in the plasma membrane and the septum is a plasma membrane. Around 2% of the plasma membrane are those H+-ATPase’s and they are of great importance for the cells to maintain their intracellular pH, ionic balance and they are needed to maintain the membrane potential which is needed for nutrient uptake. Furthermore it is known that the PMA gene is highly conserved in different plant and fungi species. The similarity in amino acid sequence is between 45-95%. For the H+-ATPase that was used in the experiment described below the similarity was used and the gene known from Aspergillus fumigatus was used.