Team:uOttawa/Description
Project Description and Inspiration
Traditional methods to study gene function and networks often employ the use of plasmids. Although this approach is acceptable when studying a small number of genes, high copy plasmids create a metabolic burden in the cell. As a result, it can be difficult to study large gene networks. To alleviate this issue, some groups have engineered low copy plasmids that behave similarly to chromosomes. Unfortunately, these plasmids cannot completely reproduce the functions of regular chromosomes, resulting in significant cell-to-cell heterogeneity in phenotypes.
Some scientists, such as Zucca and collaborators, have worked to develop plasmids that adhere to the BioBrick Standard 10 and provide a method to incorporate inserts into E. coli chromosomes. However, this system is restricted to prokaryotes, which makes it incompatible with S. cerevisiae, the simplest eukaryotic organism. Unfortunately, this leaves a large gap in genetic manipulations of the eukaryotic genome and limits the research efficiency of labs that work predominantly with S. cerevisiae.
Technical complications of synthetic biology, in addition to financial constraints, are especially problematic for young scientists. We can speak from experience: iGEM was the first opportunity for many of us to work in synthetic biology, and the learning curve was very steep.
Thus, our project endeavours to tackle the obstacles of the lack of expertise and financial constraints to promote the accessibility of synthetic biology. We create a support system that would prepare inexperienced iGEM members, or any synthetic biology amateur, for experimental success. We write easy-to-read and thorough protocols accompanied with succinct videos to train amateurs in the fundamentals of synthetic biology. We collaborate with other iGEM teams to identify the fundamental skills that must be addressed by our protocols and to eliminate common sources of errors among beginners. We invite students from various educational backgrounds and levels to test our protocols. We analyze their performance and their feedback to ameliorate our protocols.
To further facilitate cloning in eukaryotes, we develop a library of BioBrick plasmid backbones that are compatible for cloning in both E. coli and S. cerevisiae using the basic techniques explained in our protocols. The plasmid backbones adhere to the RFC 10 Standard and Type IIS Assembly and allow for the systematic and efficient cloning of a desired gene within target yeast chromosomal loci, Ade2, His3, Ade4, and Gal4 loci, and is equipped with KanMX, NatMX, Ura3, and His3 yeast-selectable markers as well as RFP to enable colorimetric selection in E. coli. This library reduces the number of steps, and the amount of troubleshooting, preceding a yeast transformation.
To circumvent the financial constraints of synthetic biology, we deliberately develop our protocols and the plasmid backbone library to reduce costs associated with the use of high-end cloning resources and kits. However, because we understand the importance of staying up-to-date with modern cloning technologies, we also venture to reduce the cost of non-traditional cloning, namely Gibson Assembly. We design BioBricks of the Gibson Assembly enzymes and provide a detailed account of the expression, extraction, and purification of the proteins. We also provide instructions for the making of a ‘home-made’ Gibson Assembly kit.