Team:ULaval/Parts
Parts Overview
As a reminder, the aim of our project is to biosynthesize adrenaline by using budding yeast. Basically, we have to give to the cell genetic information that codes for the enzymes catalyzing the biochemical reactions which transform L-tyrosine into adrenaline. This pathway is comprised of four enzymes: tyrosine hydroxylase (Th), L-DOPA decarboxylase (LDDC), dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT).
Parts design starts by finding the human protein sequences of the four genes encoding the enzymes involved in the biosynthesis of adrenaline, which were downloaded from the UniProt online database. The second step was to codon optimize the DNA sequences for our chassis, S. cerevisiae, in order for the yeast genes expression machinery to work more efficiently with the provided sequences. Indeed, yeast codon usage is different from its human counterpart. We started directly from the correct protein isoform amino acid sequence, as the initial coding DNA sequence possessed introns. The initial protein sequence is accessible on the part’s page by using the UniProt code.
We also planned to submit parts used in this project. To do that, we needed to respect RFC10 rules provided by the iGEM Registry. Once the sequence was codon-optimized for yeast, we introduced silent mutations to make the coding sequences RFC10 compatible.
As third step, we ordered the four optimized DNA sequences from Integrated DNA Technologies (IDT). As we used promoter and terminator sequences that are already in the iGEM parts bank, we decided to synthetize all the parts containing promoter, coding direct sequences (CDS) and terminator. Then, we tried to clone these sequences in yeast expression plasmids and to send them to the iGEM parts bank.
As we are manipulating human DNA sequences, we are aware of the ethical concerns of our project. We invite you to look at our Human Practices where we discussed these issues and present the public input we gathered from a survey we conducted about synthetic biology and the ethical implications of our project.
Table 1 : Team iGEM ULaval parts overview
Parts design starts by finding the human protein sequences of the four genes encoding the enzymes involved in the biosynthesis of adrenaline, which were downloaded from the UniProt online database. The second step was to codon optimize the DNA sequences for our chassis, S. cerevisiae, in order for the yeast genes expression machinery to work more efficiently with the provided sequences. Indeed, yeast codon usage is different from its human counterpart. We started directly from the correct protein isoform amino acid sequence, as the initial coding DNA sequence possessed introns. The initial protein sequence is accessible on the part’s page by using the UniProt code.
We also planned to submit parts used in this project. To do that, we needed to respect RFC10 rules provided by the iGEM Registry. Once the sequence was codon-optimized for yeast, we introduced silent mutations to make the coding sequences RFC10 compatible.
As third step, we ordered the four optimized DNA sequences from Integrated DNA Technologies (IDT). As we used promoter and terminator sequences that are already in the iGEM parts bank, we decided to synthetize all the parts containing promoter, coding direct sequences (CDS) and terminator. Then, we tried to clone these sequences in yeast expression plasmids and to send them to the iGEM parts bank.
As we are manipulating human DNA sequences, we are aware of the ethical concerns of our project. We invite you to look at our Human Practices where we discussed these issues and present the public input we gathered from a survey we conducted about synthetic biology and the ethical implications of our project.
| Code | Type | Description | Designer | Length (pb) |
|---|---|---|---|---|
| BBa_K2672000 | Coding | Human Tyrosine hydroxylase (Sc codon optimized) | François Rouleau | 1587 |
| BBa_K2672001 | Coding | Human L-DOPA decarboxylase (Sc codon optimized) | François Rouleau | 1443 |
| BBa_K2672002 | Coding | Human Dopamine beta-hydroxylase (Sc codon optimized) | François Rouleau | 1854 |
| BBa_K2672003 | Coding | Human Phenylethanolamine N-methyltransferase (Sc codon optimized) | François Rouleau | 846 |