Design

Before anything can be done in the lab, a clear goal is needed. Our goal being crocin production, we investigated the pathways for crocin production. We found out that Rhodotorula toruloides only needs 3 added reactions for crocin synthesis to be possible. Using a genome-scale metabolic model this was confirmed (see Modeling) After the decision to use R. toruloides as our crocin producing organism, the necessary transcriptional unit sequences were created and codon optimised.

Build

The build cycle included the creation of transcriptional units and transformation of R. toruloides. The transcriptional unit (TU) creation involved a DBTL cycle itself, because our methods of Golden Gate assembly (GGA) and PCR amplification needed to be optimised for high GC content sequences. After many cycles of testing possible solutions for the TU creation, the correct primers and PCR conditions were found. Before transforming R. toruloides with the final construct, which involves 3 protein coding genes and an antibiotic resistance gene, the antibiotic resistance gene needed to be tested. This is because, if our selection marker doesn’t work, selection of transformants would be impossible.

Test

After transforming R. toruloides with antibiotic resistance, the possible transformants were tested for antibiotic resistance. By using both G418 and hygromycin as the antibiotics, we achieved some colony growth only with hygromycin and its resistance as the marker. Although we saw growth on hygromycin, later testing couldn’t prove that the colonies were R. toruloides and suggested that an infection with a hygromycin resistant bacteria occurred.

Learn

2 possible things could lead to us not getting transformants - either our part sequences weren’t optimal for R. toruloides or our transformation protocol didn’t work as expected. This could be further investigated by using another transformation method - for example Agrobacterium mediated transformation which has been shown to work with this yeast. The next DTBL cycle would involve part creation for Agrobacterium mediated transformation, transformation of the yeast with Agrobacterium, and testing of the transformants for antibiotic resistance. If this cycle again would yield no successful transformants, a deeper look into codon optimization and part creation would be required.