Introduction
On this page, we will showcase our contributions to the iGEM community. In our project, we provided and optimized an efficient, convenient and universal construction strategy, and combined this strategy to develop an automated and multifunctional hardware adapted to general construction experiments. We hope that the contents of our project will help future iGEM teams to improve the efficiencies of their construction experiments, and greatly reduce the labor and time costs in the DNA assembly process.
Construction strategy
Our team took the lead in using the new DNA assembly strategy in iGEM, YeastFab, which is an efficient multigene assembly strategy. This assembly strategy can further improve the standardization degree of the elements in a DNA assembly process, reduce the complexity of operation steps, and minimize the time cost. Future iGEM teams can also adopt this strategy to complete their own assembly of multiple genes efficiently.
Part
Our team contributed to the iGEM Registry by adding multiple new parts.
Promoters: We collected more than 30 promoters of S. Cerevisiae in the Eukaryotic Promoter Database website, and used the expression strength of the mCherry, a red fluorescent protein, to calibrate the expression strength of the promoters. Promoters of different intensity play an important role in regulating downstream gene expression. Meanwhile, a large amount of data about the promoter strength are helpful to the rational combination for the expression of enzymes involved in the synthesis pathway of a target product.
CDSs: We collected eranylgeranyl diphosphate synthase TmCrtE from Taxus media, phytoene synthase PaCrtB from Pantoea agglomerans and phytoene desaturase BtCrtI from Blakeslea trispora in the heterologous lycopene Biosynthetic pathway. In addition, we have also standardized seven enzymes of mevalonate (MVA) pathways involved in the MVA pathway, such as tHMG, IDI, etc.
Terminators: We have collected 9 different terminators for standardized construction. The abundance of terminators can effectively avoid homologous recombination in the S. Cerevisiae genome. Different terminators also have distinct stabilizing effects on the mRNA produced by the transcription of a target gene
We paid attention to an existing basic component in the component library, the S. Cerevisiae promoter PGK1, and tried to improve it. As we know, an excessively long promoter sequence is not conducive to molecular experiments, DNA sequencing and bioinformatics analysis, and brings a lot of trouble to researchers. Currently, the length of the PGK1 promoter in the iGEM component library is 1497 bps. We truncated it to 1100, 800, 600, 400 and even 200 bps, and tested them using mCherry as a reporter. These experiments helped us to determine the minimal strength of the PGK1 promoter with different lengths to identify its optimal or shorter length with the same strength compared to the original promoter.
Hardware
The hardware part of our team is mainly composed of four parts -- the frame module, pipette module, robotic arm module and visual identification module. The three major operations in the construction experiment -- pipetting, constant temperature treatment and bacterium picking -- can be realized automatically by the collocation algorithms of these modules. Our hardware is versatile and cost-effective. Meanwhile, we also optimized the corresponding interactive software for the hardware part to facilitate the operation. This makes our hardware suitable for sharing with other teams in the iGEM community, and helping other teams to create their own automated construction platforms at a lower cost.
