Part:BBa_K3046015

PLEAPgfaA_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 on unknown for Aspergillus niger that is expected to have high activity in the late exponential phase and stationary 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 gfaA promoter from different Aspergillus spp., and the gene was chosen based on RNA-seq data from Aspergillus niger. [1] This version has introduced stokastic noise compared to the consensus sequence of the promoters in Aspergillus and is therefore expected to have a lower 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 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. PLEAPgfaA_2 is expected to show constitutive expression.

Figure 1: The figure shows RNA-seq data for Aspergillus niger in both exponential and stationary phase with the gfaA 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 behave relatively constitutive with a high expression.

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 894
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]