In this page the main findings and interpretations of the results of our project can be found. In our project we aimed to achieve four different goals. These four proof of concepts are summarized in Figure 1.

As we plan on feeding our GMO with ammonia extracted from the MOF, an essential part of our project is that our GMO should be able to grow in the presence of ammonia as its only nitrogen source. We therefore grew 6 different Saccharomyces strains under different ammonia concentrations to see if the presence of ammonia would affect their growth. In the end we were able to identify the best performing strains. The protocol can be found in the Experiments page.

As can be seen from Figure 2 there are 4 strains that outperformed in the experiment, those strains were chosen for further experimentation. The doubling time of the chosen strains can be seen in Table 1.

Doubling time (T_D) for each strain in different ammonia concentrations in the exponential phase.

For our second proof of concept, we wanted to demonstrate that it is possible to use Golden Gate Assembly to clone heterologous genes for alpha-amylase in Saccharomyces spp. Going from the gene of interest to the cassette plasmid expressed in Saccharomyces spp. is a long process, through it there were some checkpoints that we used to confirm the success of the cloning: GFP screening; DNA concentration measurements with a Nanodrop; basic parts sequencing; and digestion of the final cassette plasmids.

GFP screening

The empty plasmid (pRS426__ConLS'-GFPdropout-ConRE'-URA3-2micron-Kan) used in the Golden Gate cassette plasmid assemblies contains a GFP drop-out. This way, when the assembled plasmid is inserted correctly, the GFP insert will drop-out and white colonies will be formed. Whenever the plasmid was not inserted correctly the GFP drop-out will still be present and colonies will appear green when examined with UV-light (Figure 3). By this simple technique we make sure that the chosen colonies have uptaken the assembled plasmid without the need of performing sequencing or digestion experiments prior to plasmid isolation. This GFP screening was used both for assembling our part plasmids and cassette plasmids.

DNA concentrations

After purification of cassette plasmids amplified in E. coli, DNA concentrations were measured in order to assure a successful purification prior to Saccharomyces spp. transformations, results can be found in Table 2. These measurements were performed using a nanodrop spectrophotometer, measuring the DNA concentration at a spectrum of wavelengths.

DNA concentration and purity after E. coli transformation of cassette plasmid and plasmid isolation.

DNA sequencing

To ensure that our ordered G-blocks for part 3 and part 3B we made were correct, we sequenced the basic parts (4 full alpha amylases and 4 alpha amylases without the native secretion peptide) assembled in the entry vector (more on this on the Engineering page). pYTK001 (entry vector) with Part 3 and 3b was sequenced using two primers flanking the gene, the vector is used to compose the cassettes plasmids in further experiments. All sequencing results are summarized in Table 3.

Sequencing results from all the parts.

Constructs digestion

We used the restriction enzyme BsmBI to check for the correct ligation of the different constructs of the library. There are two sites for the enzyme BsmBI in the vector employed (pRS246) for our constructs, the sites are in part 1 and part 5, on both sides of the insert of interest (Figure 4). The use of BsmBI allows us to check for the size (Table 4) of the construct and assess the success of the assembly. The results of the digestion can be found in Figure 5.

Expected sizes of the different constructs of the library. The elements of each sample can be found in the pages of the Parts plasmids.

For our third proof of concept, we wanted to show that our GMO does indeed show alpha-amylase activity after taking up our construct. We aimed to measure the Alpha amylase production of 64 samples listed at our Engineering page. This was tested with two main experiments. The principal one being an alpha amylase kit assay used to perform high throughput experiments and obtain quantitative data, needed for our model (see Model page). Additionally, a qualitative assay was employed to confirm the production of alpha amylase in some of the samples. To understand more how this assay works, see the Experiments page.

Alpha-amylase starch breakdown

Functionality of alpha amylase can qualitatively be proved by observing the breaking down of starch. The ability to break down starch is only available when amylase is produced, which in the case of S. cerevisiae and S. paradoxus doesn’t happen natively (Figure 6e). However, the assembled constructs should be able to break down starch. In order to confirm this, selective media plates without uracil (specific for the assembled constructs) were created with the addition of 1% starch. Several samples were cultured ON and treated before being plated on the starch plates, as described in the notebook (wk 39), after which pictures were taken (Figure 6) of the with/without iodine treated starch plates. Samples tested with this assay can be found in Table 5.

Composition of samples that were tested by the Halo assay.