Team:Johns Hopkins/YT/Over

From 2011.igem.org

VitaYeast - Johns Hopkins University, iGEM 2011

Overview of Yeast Toolkit
Combinatorial Yeast Expression Cassette Library
Forward Constitutive Promoter.png
We are creating a library of yeast promoters and 3'UTRs that are fully compatible with the BioBrick standard. The sequences come from the upstream and downstream region of 12 yeast genes, and are associated with varying levels of expression strength: 4 for strong expression, 4 for medium expression, and 4 for weak expression.

In addition to our BioBrick compatible promoters and 3'UTRs, we have also given the same sequences designed overhangs with BsaI sites to facilitate Golden Gate assembly, which we are using in our violacein projects. The Golden Gate assembly method has advantages over BioBrick assembly in that multiple pieces can be ligated together at once with no scar.

Yeast Shuttle Vector Library

Using Sikorski and Christianson et al.'s pRS 400-series shuttle vectors as a base, we created a vector platform that is fully compatible with the BioBrick Standard Assembly. This involved site-directed mutagenesis to remove unique restriction sites from within the vector as well as a multiple cloning site substitution. The end product is a vector with a BioBrick multiple cloning site for use with Standard Assembly. Because of the various needs of synthetic biological systems in terms of artificial selection, our vector library contains an array of selectable markers, including histidine, leucine, tryptophan and uracil biosynthetic genes for use in various knockout strains of S. cerevisiae. Each respective marker is available in three plasmid types: integrating, CEN/ARS, and 2micron high copy number vectors. The vectors can be propogated in E. coli with an ampicillin resistance selection as well as transformed into S. cerevisiae.

Violacein Pigment: A Novel Yeast Reporter
Violacein (Source)

Violacein purple pigment production serves as a simple way to test a multiple gene pathway (such as vitamin production) in an expression vector or neochromosome. If the gene construct is transformed and the organism becomes purple, the system is successful. As our main project is in yeast and since purple bacteria was achieved by Cambridge iGEM 2009, we focused on producing violacein in the eukaryotic S. cerevisiae yeast. We chose violacein specifically as a Boeke Lab rotation student had begun to engineer the violacein pathway in yeast.