Part:BBa_K3046013

PLEAPmstA_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 very strong constitutive promoter for Aspergillus niger that has especially high activity in the exponential growth phase.

Characterization

This is a synthetic constitutive promoter, created as part of the LEAP (Library of Engineered Aspergillus Promoters) project. It is based on the mstA 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 a microbioreactor (BioLector, m2p-labs)
The microtiter scale cultures were made by inoculating 10⁷ spores in 1.5 mL minimal media, and cultures were grown at 30 °C with mixing at 1000 rpm for 78 hours. Both biomass (measured in light scattering units) and fluorescence (at Ex/Emi wavelengths 580 nm/625 nm) was measured continuously.

The predicted behavior of this promoter, is described by the model, which is summarized by the figure below. PLEAPmstA_1 is expected to show constitutive expression with high promoter strength.

Figure 1: The figure shows RNA-seq data for Aspergillus niger in both exponential and stationary phase with the mstA 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 consensus promoter should be relatively constitutive with a high expression with this variant being constitutively active, but weaker.

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 BamHI site found at 707
  Illegal XhoI site found at 532
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]