Part:BBa_K3046001

PLEAPglaA_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 strong promoter for Aspergillus niger that has high activity in the exponential 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.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 multiple scales using a confocal microscope, a microbioreactor (BioLector, m2p-labs) for microtiter scale, in shake flasks for medium scale and in a 1 liter bioreactor for large 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.
In the medium scale, cultures were grown in 200 mL minimal media in 500 mL shake flasks with baffles at 30 °C and 250 rpm. Here samples were taken with regular intervals.
The promoter was also tested at 1 liter bioreactor scale and grown in 1 L minimal media, pH 5, 800 rpm, and samples were taken with regular intervals. Additionally, the off-gas was measured for CO₂ content to determine the growth.
The data for the shake flask- and bioreactor-scale experiments wea generated by purifying protein from the sampled biomass and computing the TexasRed equivalent fluorescence per protein. A Bradford assay was used to determine the protein concentration after extraction, and fluorescence was measured in a plate reader at Ex/Emi wavelengths 580 nm/625 nm.

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 microscopy, the samples were analyzed under a confocal microscope for red fluorescence showing the following. We see that mCherry is produced in the fungi which means that the promoter works.

Figure 2: Pictures from confocal microscopy of A. niger producing mCherry using the promoter PLEAPglaA_2.

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 3: 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 it is seen that the promoter starts expression of mCherry around 30 hours.

In the medium scale, the promoters were analyzed in duplicate and the TexasRed equivalent fluorescence per protein was graphed, as seen below.

Figure 4: This figure shows 5 different promoters , run in two duplicates. The top row is replicate one and the bottom row is replicate two. From left to right, the columns are: Negative control, Positive control, PLAEPglaA_1, PLEAPglaA_2, PLEAPsonB_1, and PLEAPunk_1. The y-axis is showing mmol TexasRed/µg protein, x-axis is showing time in hours. Here, a distinct difference from the negative control is seen for PLEAPglaA_2.

Lastly, the promoter was analyzed in a 1 liter bioreactor.

Figure 5: Graphs show data from 1 L bioreactors. The top row shows equivalents of TexasRed per protein and the bottom row shows accumulated CO₂, which is correlated to biomass growth. From left to right is seen following samples : Negative control, Positive control, PLEAPsonB_1 run 2, PLEAPsonB_1 run 1, and PLEAPglaA_2. Here it can be seen that PLEAPglaA_2 is distinct from the negative control with regards to fluorescence.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 50
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]