Regulatory

Part:BBa_K3046010

Designed by: Marcus Medom Ryding, Mikkel Rasmus Hansen   Group: iGEM19_DTU-Denmark   (2019-10-16)


PLEAPglaA_1

This is a synthetic constitutive promoter, created as part of the LEAP (Library of Engineered Aspergillus Promoters) project

Usage and Biology

This is a promoter for Aspergillus niger of unknown strength that is expected to have high expression in the exponential growth phase.

Characterization

This is a synthetic exponential phase promoter, created as part of the LEAP (Library of Engineered Aspergillus Promoters) project. It is based on the glaA promoter from different Aspergillus spp., and the gene was chosen based on RNA-seq data from Aspergillus niger. [1] The consensus promoter was expected to have really high expression. This version has “noise” added to the sequence to support a hypothesis that it was possible to turn down expression of the promoter.
This promoter was characterised using an mCherry test device,BBa_K3046009, inserted into an AMA1-based test plasmid, BBa_K3046021, and characterisation was done in a microbioreactor (BioLector, m2p-labs) for microtiter scale.

The predicted behavior of the consensus promoter, as described by the model, is summarized by the figure below. PLEAPglaA_2 is expected to show a much lower expression, but maintain the same characteristics with regards to growth phase dependency.

Figure 1: The figure shows RNA-seq data for Aspergillus niger in both exponential and stationary phase with the glaA gene marked in red. The x-axis is the promoter activity in the stationary phase and the y-axis is the promoter activity in the exponential phase, both axes are depicted on a log scale. Here we see that this promoter should be more active in the exponential phase than in the stationary phase.

For the microtiter scale, the promoter was evaluated in a biolector, producing the following results with regards to growth, fluorescence and dynamic promoter activity.
Figure 2: When analyzed in the microtiter scale on a biolector, the red fluorescence and biomass has been measured and the Dynamic Promoter Activity has been calculated. On the graph Dynamic Promoter Activity (DPA) is green, the biomass is measured in Light Scattering Units (LSU) is in blue, and red fluorescence (RFP) is shown in red. The graphs have been normalized for LSU. Here we see no appreciable promoter activity


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 342
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 354
    Illegal BamHI site found at 165
    Illegal XhoI site found at 599
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
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