Difference between revisions of "Team:DTU-Denmark/Results"

We set out to design a piece of software that could be used to create synthetic constitutive promoters, and we wanted to build a promoter library in Aspergillus niger as a proof of concept to demonstrate that the software worked on a fundamental level. By performing different experiments during the summer, we have been able to demonstrate the following: From our software “proHMMoter”, we are able to design promoters that are functional, while also being domesticated to be in compliance with common cloning standards. We have demonstrated that the promoters can work when cultivating in several different scales

• The proHMMoter is able to produce promoters that have different dynamics, in a manner that can be predicted by modelling based on RNA-seq data
• proHMMoter can additionally be used to create promoters of varied strength

Library construction

We were able to integrate all our tested promoters into our promoter MoClo test device. As mentioned on our vector design page, the test device is designed with a screening mechanism for Golden Gate cloning that should result in red colonies if the plasmid has been re-ligated, and white colonies if the placeholder promoter has been replaced in favor of an insert promoter. As seen in figure 1, the system appears to work, as we can see mCherry being expressed by the vast majority of cells when the placeholder promoter has not been removed. When inserting the promoters, we can conversely see that the vast majority of colonies turn white, while a subset of the transformants keep expressing mCherry. Sanger sequencing confirmed that the white colonies contained the inserted synthetic promoters as expected.

Figure 1 shows the transformants before and after the insertion of the MoClo promoter test plasmid. The left part of the figure shows that there is production of mCherry if the MoClo promoter is not inserted, whereas to the right, it can be seen that the transformants with the inserted MoClo promoters stops producing mCherry.

Qualitative results to demonstrate working promoters

After confirming that our test device worked as expected, we wanted to verify that we were able to express mCherry from our promoters. To do this, we inserted our promoter test device into the autonomously replicating AMA1-zing_pyrG plasmid, and used the plasmid to transform our A. niger ATCC1015 strain. From the fungal strains containing our test plasmids, we did a few short experiments to get some qualitative data on whether the promoters worked or not. We inoculated 10 mL cultures of minimal media in 50 mL ventilated falcon tubes, and incubated them for 80 hours at 37 C at 150 rpm. As seen in figure 2, the promoters were able to produce mCherry in sufficient quantities for it to be visible in the culture compared to the negative control.

When looking at the culture samples through a confocal microscope, we are similarly able to see a clear difference in fluorescence between the sample and the negative control. The fluorescent images below of the mCherry expressed by our fungal strains were taken by a confocal microscope. To the left is the fluorescence of our synthetic promoters, and to the right is the fluorescence of the negative control.

BioLector data

Microplate scale experiments was carried out by using a BioLector as mentioned. Each well was inoculated with 10E7 spores in 1,5 mL minimal media. For positive control, we used a PH4H3 histone promoter expressing mCherry in a reporter device that was genome integrated in A. nidulans. For negative control, A. niger transformed with an AMA1 plasmid with no mCherry device was used.

The graphs above display data collected from BioLector cultivations of a range of our synthetic promoters. The blue line represents the positive control, and the red line represents the negative control - and what is very interesting are the lines lying between them. These represent working synthetic promoters of varying strengths, and demonstrate our software’s ability to predict and create a synthetic promoter ladder, i.e. promoters of varying strengths, for a given organism. The other graph displays light scattering units (LSU) as a function of time, and represents the growth of the different strains. From the LSU, it is evident that the different strains grow with very variable rates - this underlines the need to correct for biomass/cell growth when analysing the fluorescence data from the BioLector. This has been done for the datasets available both on our Demonstrate page, our parts pages on the Registry, and in our “pick-your-promoter” widget.

Shakeflask data

We tested three of the LEAP promoters in 200 mL cultures in Erlenmeyer flasks, to see how the promoters would behave when scaling up from BioLector wells. Normalised fluorescence data from all the shake flasks can be seen in figure 6

From the figure above it can be observed that the PLEAPglaA_1 and PLEAPsonB_1 promoters expresses relatively low amount of protein, whereas the PLEAPglaA_2 and PLEAPunk_1 promoters expresses a higher amount when adjusting for total protein expression. Our data from figure 6 suggest that PLEAPglaA_2 has the highest promoter activity.

Based on these results, we believe that we have succeeded in creating a promoter library. For more details, check our in-depth measurements on the demonstrate page page.

Further results from side projects

Besides our main mission, which has been to demonstrate that our proHMMoter software works by building a promoter library, we have also worked on a lot of smaller projects that are all related to the goal of making synthetic biology in filamentous fungi easier and more accessible in the future.
First of all, we have collected some qualitatively data for PtrpC - an Aspergillus spp. promoter that are already present in the Registry of Standard Biological Parts, but a part that did not have any quantitative data. For more info on the characterisation we did, look at our characterization page.
Furthermore, we have succeeded in creating a prototype design for genome integrating promoters into the Aspergillus niger genome. pPEA2P1 (BBa_K3046031) was shown to express eGFP as expected, and the successful transformants was subsequently used for the PtrpC characterisation mentioned above.