Difference between revisions of "Part:BBa K1021007"

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<b>GFP expression in <i>Aspergillus niger</i> under PtrpC control</b>
 
<b>GFP expression in <i>Aspergillus niger</i> under PtrpC control</b>
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The characterisation of the PtrpC promoter was done by integrating the promoter into the plasmid, as described at the <a href="https://2019.igem.org/Team:DTU-Denmark/Design_Plasmid" target="_blank">Design page</a>.
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The characterisation of the PtrpC promoter was done by integrating the promoter into the plasmid, as described at the <a href="https://2019.igem.org/Team:DTU-Denmark/Design_Plasmid" target="_blank">Design page.</a> <br>
  
 
The promoter was characterised by two separate experiments.  
 
The promoter was characterised by two separate experiments.  
  
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In the first experiment, the PtrpC strain was cultivated in quadruplicates in 50 mL breathable falcon tubes in 10 mL minimal media. A negative control consisting of the parental strain was included. The strains were cultivated for 80 h. at 37C with shaking at 150 rpm. The culture was then stored at 4C, and subsequently protein was extracted and florescence measured as described on the <a href="https://2019.igem.org/Team:DTU-Denmark/Experiments" target="_blank"> Experiments </a>page. The resulting data can be found below.
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In the first experiment, the PtrpC strain was cultivated in quadruplicates in 50 mL breathable falcon tubes in 10 mL minimal media. A negative control consisting of the parental strain was included. The strains were cultivated for 80 h. at 37C with shaking at 150 rpm. The culture was then stored at 4C, and subsequently protein was extracted and florescence measured as described on the <a href="https://2019.igem.org/Team:DTU-Denmark/Experiments" target="_blank"> Experiments page.</a> <br>
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The resulting data can be found below.
  
 
<img src="https://static.igem.org/mediawiki/parts/9/9f/T--DTU-Denmark--GFP_PtrpC_10mL.png" style="width: 80%; padding: 15px;" >
 
<img src="https://static.igem.org/mediawiki/parts/9/9f/T--DTU-Denmark--GFP_PtrpC_10mL.png" style="width: 80%; padding: 15px;" >

Revision as of 21:43, 21 October 2019

PtrpC

PtrpC is a strong constitutive promoter from Aspergillus nidulans's tryptophan biosynthesis pathways. It was isolated using a fungal transformation vector and constructed into the pSB1C3 backbone for continuous homologous and heterologous gene expression in fungal chassis.

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


To test the activity of the trpC promoter, a composite part was created with GFP downstream (BBa_K1021023). This part was transformed into the ascomycete fungi Cochliobolus heterostrophus. Results indicated that the trpC promoter provided GFP expression within this fungal chassis.

Cochliobolus_fluorescence_composite.png

Characterization

DTU-Denmark 2019 characterization The DTU-Denmark 2019 team has characterized the PtrpC promoter in Aspergillus niger. Our characterization experiments were done in two different scales: 1.5 ml wells in a microbioreactor (BioLector), and 10 mL cultures in specialized 50 mL falcon tubes.

GFP expression in Aspergillus niger under PtrpC control The characterisation of the PtrpC promoter was done by integrating the promoter into the plasmid, as described at the Design page.
The promoter was characterised by two separate experiments. In the first experiment, the PtrpC strain was cultivated in quadruplicates in 50 mL breathable falcon tubes in 10 mL minimal media. A negative control consisting of the parental strain was included. The strains were cultivated for 80 h. at 37C with shaking at 150 rpm. The culture was then stored at 4C, and subsequently protein was extracted and florescence measured as described on the Experiments page.
The resulting data can be found below.

Figure 1: The figure above clearly shows the many fold increase in florescence of the PtrpC promoter strain replicates compared to the negative control.
In the second experiment, the expression of GFP over time from PtrpC was measured using a micro-bioreactor (Biolector, m2p-labs). The trpC promoter was tested in seven different wells in the BioLector by measurements of fluorescence and biomass every 5 minutes for the 85 hours the characterization experiment was running. The concentration of the GFP-FU, was calculated by a standard curve, that was constructed from the results form the biolecter. The GFP-FU equivalent fluorescence is illustrated in the figure below.
Figure 2A: The figure above displays the GFP florescence measured in 7 replicates from the Biolector run.
The figure below illustrates the same data as a function of light scattering units (biomass).
Figure 2B: The figure above displays the GFP florescence as a function of light scattering units, an approximation for biomass, measured in 7 replicates from the Biolector run.