Difference between revisions of "Part:BBa K1021007"

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===Characterization===
 
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<b>DTU-Denmark 2019 characterization</b>
 
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The DTU-Denmark 2019 team has characterized the PtrpC promoter in <i>Aspergillus niger</i>. 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.   
 
  
 
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<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 DTU-Denmark 2019 wiki <a href="https://2019.igem.org/Team:DTU-Denmark/Design_Plasmid" target="_blank">page.</a> <br>
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<span class="mw-headline" id="DTU_2019s_Characterization">DTU-Denmark 2019's Characterization</span>
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The promoter was characterised by two separate experiments.  
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The DTU-Denmark 2019 team has characterized the PtrpC promoter in <i>Aspergillus niger</i>. 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.  
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<br><br>
  
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<h3>GFP expression in <i>Aspergillus niger</i> under PtrpC control</h3>
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The characterisation of the PtrpC promoter was done by integrating the promoter into the plasmid, as described at the DTU-Denmark 2019 wiki <a href="https://2019.igem.org/Team:DTU-Denmark/Design_Plasmid" target="_blank">page.</a> <br>
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<br>
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The promoter was characterised by two separate experiments.
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<br><br>
 
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 37°C with shaking at 150 rpm. The culture was then stored at 4°C, and subsequently protein was extracted and fluorescence measured as described on the DTU-Denmark 2019 wiki <a href="https://2019.igem.org/Team:DTU-Denmark/Experiments" target="_blank">page.</a> <br>
 
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 37°C with shaking at 150 rpm. The culture was then stored at 4°C, and subsequently protein was extracted and fluorescence measured as described on the DTU-Denmark 2019 wiki <a href="https://2019.igem.org/Team:DTU-Denmark/Experiments" target="_blank">page.</a> <br>
  

Latest revision as of 00:56, 22 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


DTU-Denmark 2019's 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 DTU-Denmark 2019 wiki 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 37°C with shaking at 150 rpm. The culture was then stored at 4°C, and subsequently protein was extracted and fluorescence measured as described on the DTU-Denmark 2019 wiki page.
The resulting data can be found below.
Figure 1: The figure above clearly shows the many fold increase in fluorescence 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 (GFP-fluorescence units), 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 fluorescence measured in 7 replicates from the Biolector run. The Figure shows the expression of GFP-FU, where it enters the experimental stage, after 5 hours, and peaks after 40 hours, with an approximate concentration of 14 nM. The expression stabilizes at approximately 10 nM.
The figure below illustrates the same data as a function of light scattering units (biomass).
Figure 2B: The figure above displays the GFP fluorescence as a function of light scattering units, an approximation for biomass, measured in 7 replicates from the Biolector run. The Figure shows the expression of GFP-FU per biomass. It increases for the first ten hours to approximately 0.14 nM GFP-FU/biomass. It declines afterwards and is stabilized at a concentration of around 0.05 nM GFP-FU/biomass.