Part:BBa_K3046002
PLEAPsonB_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 medium strength constitutive promoter for Aspergillus niger.
Characterization
This is a synthetic constitutive promoter, created as part of the LEAP (Library of Engineered Aspergillus Promoters) project. It is based on the sonB 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 sonB promoters in Aspergillus and is expected to have a medium strength 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 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 this promoter, is described by the model, which is summarized by the figure below. PLEAPsonB_1 is expected to show constitutive expression with medium promoter strength.
For microscopy, the samples were analyzed under a confocal microscope for red fluorescence as seen in the following picture. We see that mCherry is clearly produced, which shows that the promoter works in A. niger.
For the microtiter scale, the promoter was evaluated in a biolector, producing the following results with regards to growth, fluorescence and dynamic promoter activity.
In the medium scale, the promoters were analyzed in duplicate and the TexasRed equivalent fluorescence per protein was graphed, as seen below.
Lastly, the promoter was analyzed in a 1 liter bioreactor.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 602
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 694
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 343
Illegal BglII site found at 654 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 602
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 602
- 1000COMPATIBLE WITH RFC[1000]
//promoter
//regulation/constitutive
//rnap/eukaryote
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