Difference between revisions of "Part:BBa K3046005"

Latest revision as of 03:17, 14 December 2019

PLEAPmstA_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 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] This version is a consensus sequence of the mstA promoters in Aspergillus and is expected to have a high constitutive 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 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 promoter should be 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 it is seen that the promoter has a high dynamic promoter activity that peaks around 20 hours and subsequently evens out after 40 hours.

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 61
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 470
1000
COMPATIBLE WITH RFC[1000]

@@ Line 18: / Line 18: @@
 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.
 <br>
-<img src="https://static.igem.org/mediawiki/parts/2/21/T--DTU-Denmark--RNASEQmstA_1.png" style="width: 80%; padding: 15px;" >
+<img src="https://2019.igem.org/wiki/images/thumb/2/25/T--DTU-Denmark--RNA_MstA.png/650px-T--DTU-Denmark--RNA_MstA.png" style="width: 80%; padding: 15px;" >
 <figure>
 <figcaption> Figure 1: The figure shows RNA-seq data for <i>Aspergillus niger</i> 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 promoter should be relatively constitutive with a high expression.