Difference between revisions of "Part:BBa K3046003"

Revision as of 17:26, 21 October 2019

PLEAPgpdA_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 strong constitutive promoter for Aspergillus niger that has especially high activity in the lag 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 gpdA 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 promoters in Aspergillus and is expected to have a high 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) 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.

Figure 1: The figure shows RNA-seq data for Aspergillus niger in both exponential and stationary phase with the gpdA 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 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 it is seen that the promoter has a high dynamic activity, starting around 10 hours and peaking around 20 hours, after which it levels out and becomes stable at 40 hours. .

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 106
Illegal AgeI site found at 35
1000
COMPATIBLE WITH RFC[1000]

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 ===Characterization===
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-This is a synthetic constitutive promoter, created as part of the LEAP (Library of Engineered Aspergillus Promoters) project. It is based on the gpdA promoter from different <i>Aspergillus</i> spp., and the gene was chosen based on RNA-seq data from <i>Aspergillus niger</i>. This version is a consensus sequence of the promoters in <i>Aspergillus</i> and is expected to have a high expression.
+This is a synthetic constitutive promoter, created as part of the LEAP (Library of Engineered Aspergillus Promoters) project. It is based on the gpdA promoter from different <i>Aspergillus</i> spp., and the gene was chosen based on RNA-seq data from <i>Aspergillus niger</i>. [1] This version is a consensus sequence of the promoters in <i>Aspergillus</i> and is expected to have a high expression.
 <br><br>
 This promoter was characterised using an mCherry test device,<a href=”https://parts.igem.org/wiki/index.php?title=Part:BBa_K3046009” target=”_blank”>BBa_K3046009</a>, inserted into an AMA1-based test plasmid, <a href=”https://parts.igem.org/wiki/index.php?title=Part:BBa_K3046021” target=”_blank_”>BBa_K3046021</a>, and characterisation was done a microbioreactor (BioLector, m2p-labs) for microtiter scale.