Autonomously replicating plasmids as a transient expression tool in Aspergillus niger

Ref ID: 18528

Author:

Ursula Kiesswetter,
Marzena Blumhoff,
Matthias Steiger,
Christine Sanystra,
Diethard Mattanovich,
Michael Sauer

Author address:

Austrian Centre of Industrial Biotechnology (ACIB GmbH),  
School of Bioengineering, University of Applied
Sciences FH-Campus Wien,
BOKU University of Natural Resources and Life Sciences

Full conference title:

11 th European Conference on Fungal Genetics

Abstract:

The possibility of having an autonomously replicating plasmid for filamentous fungi broadens the horizon of
genetic engineering. Tools like recombinases or reporter genes can easily be inserted to cells and just as well be
expelled when taking away the selective pressure. Such a system can be used as a transient expression tool for
genetic engineering purposes in Aspergillus niger.
AMA1 is a 6.1 kb DNA fragment that allows extrachromosomal replication in filamentous fungi and strongly
enhances the transformation efficiency. However, plasmid construction is hindered by the two palindromic,
inverted sequences that flank the 0.3 kb central region of the AMA1 fragment. Hints in literature led to the
assumption that the sequence can be shortened without loosing the positive influence on transformation
efficiency.
In this study we characterized different plasmids carrying shortened fragments of AMA1. The transformation
efficiencies as well as the plasmid stabilities were analyzed. The conducted experiments demonstrate that only
one of the palindromic sequences together with the central region of AMA1 are necessary for autonomous
replication in Aspergillus niger. Further shortening led to a drastic decline of transformation efficiency. Plasmids
are lost at the latest in the 3rd generation when the spores are cultivated without antibiotic pressure. On the other
hand, integration of the plasmids seems to be possible if antibiotic pressure is sustained.

Abstract Number: PR8.41

Conference Year: 2012

Link to conference website: http://www.ecfg.info/images/Abstract_Book_Electronic.pdf

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