These two parts are gene coding sequences, one encodes egt1 that from Neurospora crassa, a filamentous fungus that produces ergothioneine, another encodes egt2 that from ergot fungus Claviceps purpurea, the first strain that was found to be capable of producing ergothioneine.

Egt1 is a nonheme iron enzyme that could simultaneously catalyze two reactions, including the methylation of histidine to hecynine and the conversion of hecynine to hercynylcysteine sulfoxide. After that, another enzyme called Egt2, hercynylcysteine S-oxide lyase, will combine with hercynylcysteine sulfoxide to form EGT.

These two parts are the gene expression cassettes corresponding to the above two genes respectively. The expression cassettes both carrying a promoter, gene coding sequence and a terminator.

In order to enable the engineered strain to produce ergothioneine in a more sustainable way, we chose the constitutive promoter to introduce the expression of the two heterologous genes. According to literature references and existing resources in our laboratory, we finally chose apply pTEF1 and pADH1 to our work, they are commonly used for metabolic engineering of yeast, more importantly, both of them could provide relatively high expression values of genes, which means we could get more production of ergothioneine in the end.

Transcription terminators play a role in terminating the progress of gene transcription, and are thus essential elements in the gene expression. For both gene expression cassettes, we chose to apply tCYC to our work.

Here, these two gene expression cassettes were added with about 0.5kb homology arms on both sides when they were used as the linear integration fragments.

For better use by future iGEM teams, here are some tips. During the experiment, we found that some regions of the promoter sequence templates are rich in GC residues, which tend to fold into complex secondary structures and might not melt during the annealing phase of the PCR cycle. Also, the primers used to amplify GC-rich regions often have a high capacity to form self- and cross-dimers and a strong tendency to fold into stem-loop structures that can impede the progress of the DNA polymerase along the template molecule. Therefore, instead of PCR method, we obtained the gene expression cassettes by enzyme digestion.

van der Hoek, S. A., B. Darbani, K. E. Zugaj, B. K. Prabhala, M. B. Biron, M. Randelovic, J. B. Medina, D. B. Kell and I. Borodina (2019). "Engineering the Yeast Saccharomyces cerevisiae for the Production of L-(+)-Ergothioneine." Frontiers in Bioengineering and Biotechnology 7.