Team:DTU-Denmark/Design Plasmid
Vector Design
Creating a promoter test platform for A. niger
As there were no suitable devices in the iGEM registry for testing promoters in filamentous fungi, our team had to develop our own test platform for easy cloning and characterization of promoters. Our solution was simple: We built a new reporter device by placing an exchangeable placeholder promoter upstream of an ORF expressing a fluorescent protein. We elected to use mCherry as our reporter protein because it is visible to the naked eye in E. coli and chemical standards exist, which will ease comparisons between plate readers and labs. Our choice of mCherry is described in greater detail on the Measurement design page.
Using the new iGEM Type IIs RFC[1000] standard, we ensured that a placeholder promoter would be able to be cut out from the test device using BsaI, which would leave fusion sequences on the test device strands that would be compatible with level 0 MoClo promoter+5’UTR parts.
The whole test device is flanked by two of the Unique Nucleotide Sequences (UNSs), originally identified by Torella et. al. 2014 [2]. As the name suggests, these nucleotide sequences have been selected on the basis of being unique, i.e. these sequences do not have a direct match anywhere in the E.coli genome. When using E.coli as a host organism for constructing synthetic circuits, these UNSs can therefore be used for Gibson cloning or other kinds of homology-based cloning methods. Alternatively, the test device can be flanked by SapI sites, making the whole transcriptional unit of the test device be able to be integrated into any even plasmid when using the loop system. [1]
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