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.