Team:DTU-Denmark/Results

Results

All iGEM projects generate a lot of awesome results, and we are not an exception!

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 modeling based on RNA-seq data
  • proHMMoter can additionally be used to create promoters of varied strength

Pick your Promoter

An03g06550 (BBa_K3046001)

Alias: PLEAPglaA_2

glaA encodes a secreted glucoamylase (1,4-alpha-D-glucan glucohydrolase), and is mostly expressed at the edge of the colonies. Furthermore, it is repressed by xylose and induced by maltose.

Versions: 1 naturalistic & 1 synthetically enhanced

RNA seq data derived from [1]

When analyzed in the microtiter scale on a biolector, the red fluorescence and biomass have 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

An04g05630 (BBa_K3046002)

Alias: PLEAPsonB_1

The specific function of sonB_1 in unknown, but it’s orthologs are associated with cellular response to DNA damage and regulation of the mitotic cell cycle.

Versions: 1 naturalistic & 1 synthetically enhanced

RNA seq data derived from [1]

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

An16g01830 (BBa_K3046003)

Alias: PLEAPgpdA_1

gpdA encodes a Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is an enzyme that is involved in glycolysis, and, according to new research, it may also be involved with initiation of apoptosis and vesicle shuttling.

Versions: 1 naturalistic & 1 synthetically enhanced

RNA seq data derived from [1]

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

An16g01830 (BBa_K3046004)

Alias: PLEAPgpdA_2

gpdA encodes a Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is an enzyme that is involved in glycolysis, and, according to new research, it may also be involved with initiation of apoptosis and vesicle shuttling.

Versions: 1 naturalistic & 1 synthetically enhanced

RNA seq data derived from [1]

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

An12g07450 (BBa_K3046005

Alias: PLEAPmstA_1

mstA encodes a high-affinity sugar transporter and has been shown to be expressed when A. niger starts to run out of sugar. It doesn't care about the age/phase of the culture, only about sugar

Special Ability: sensitive sugar sensor, turns on when sugar is limited

Versions: 1 naturalistic & 1 synthetically enhanced

RNA seq data derived from [1]

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

An07g08210 (BBa_K3046006)

Alias: PLEAPunk_1

This gene has not been characterized in Aspergillus niger yet, but in other aspergilli, it seems to be involved in signal transduction along actin filaments by activating kinases.

Versions: 1 naturalistic & 1 synthetically enhanced

RNA seq data derived from [1]

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

An18g06820 (BBa_K3046007)

Alias: PLEAPgfaA_1

This gene encodes a gene called putative glutamine:fructose-6-phosphate amidotransferase (which is a mouthful) and is involved with the synthesis of the chitin that makes up the cell wall of fungi.

Versions: 1 naturalistic & 1 synthetically enhanced

RNA seq data derived from [1]

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

An08g09880 (BBa_K3046008)

Alias: PLEAPhfbD_1

The hfbD gene supposedly codes for a hydrophobin gene that is active in the dormant conidia of Aspergillus niger. This gene has a special place in our hearts as it’s expression pattern is wonderfully specific.

Versions: 1 naturalistic & 1 synthetically enhanced

RNA seq data derived from [1]

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

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 keeps expressing mCherry. Sanger sequencing confirmed that the white colonies contained the inserted synthetic promoters as expected.

Two agar plates can be seen: one with many red E. coli colonies and onw with white.
Fig. 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 a 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 stop 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.

Six falcon tubes, each containing a fungal sample with different promoters expressing mCherry
Fig. 2: To the left is the negative control, consisting of A. niger ATCC1015 with no test device transformed. The five tubes on the right all transformants containing the fungal promoter test device, each with a different synthetic promoter inserted into the test device.
This figure was taken on a confocal microscope and shows a vast net of mycelia that is expressing mCherry and fluorescing brightly red
Fig. 3: Confocal microscopy with excitation at 580 nm. mCherry is produced from one of the tubes seen in figure 2.
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.
This figure was taken on a confocal microscope and shows a vast net of mycelia that is expressing mCherry and fluorescing brightly red
Fig. 3: Confocal microscopy with excitation at 580 nm. mCherry is produced from one of the tubes seen in figure 2.

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.

This figure shows fluorescence measurements from the biolector where many graphs are plottet at once. We see that many promoters are producing more mCherry than the negative
Fig. 4: When plotting the fluorescence at Emm/Exi = 580nm/625 nm, it can be observed that at least 7 of the synthetic promoters tested in this BioLector run was expressing mCherry at a level between the negative and positive control.
This figure shows biomass measurements from the biolector where many graphs are plottet at once. We see that many cultures are growing similarity, most notably the positive and negative having similar growth
Fig. 5: When plotting the Light Scattering Units (LSU), and using it as a proxy for Biomass, it becomes apparent that the growth rates vary a lot between the different mCherry-producing strains.

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 analyzing 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. Normalized fluorescence data from all the shake flasks can be seen in figure 6

This figure shows fluorescence measurements from the shake flask experiments where five promoter were tested in duplicated.
Fig. 6: Expressed fluorescent protein per total protein over time.

From the figure above it can be observed that the PLEAPglaA_1 and PLEAPsonB_1 promoters express a relatively low amount of protein, whereas the PLEAPglaA_2 and PLEAPunk_1 promoters express 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 qualitative data for PtrpC - an Aspergillus spp. promoter that is 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 were subsequently used for the PtrpC characterization mentioned above.

The logos of our three biggest supporters, DTU Blue Dot, Novo Nordisk fonden and Otto Mønsted fonden The logos of all of our sponsors, DTU, BioNordica, Eurofins Genomics, Qiagen, NEB New England biolabs, IDT Integrated DNA technologies and Twist bioscience