Regulatory

Part:BBa_K3046012

Designed by: Marcus Medom Ryding   Group: iGEM19_DTU-Denmark   (2019-10-16)


PLEAPsonB_2

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 constitutive expression.

Characterization

This is a synthetic constitutive promoter, created as part of the LEAP (Library of Engineered Aspergillus Promoters) project. It is based on the sonB promoter from different Aspergillus spp., and the gene was chosen based on RNA-seq data from Aspergillus niger. [1] This version is a consensus sequence of the sonB promoters in Aspergillus and is expected to have a medium strength expression.
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 the promoter should be relatively constitutive.

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
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 159
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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