Team:BrownTwo/Protocols/bbyeastchrom

Moving Biobrick Constructs onto Yeast Chromosomes

A New Standard for Biobrick Construction

The Registry includes a growing number of parts for use in yeast, but there currently exist no BBa compatible yeast shuttle vectors. In our work, we have chosen to utilize Sikorski yeast vectors to integrate BBa parts into yeast chromosomes. [1] With genomic insertion, each transformation is stable, being reproduced with the rest of the chromosome. Additionally, exactly one copy of each insertion is present in transformed strains, allowing for precise control of the relative expression levels of multiple elements. The Sikorski vectors can be transformed into E. coli for construction and propagation. There exist four integrative vectors in the pRS30x family, each integrating at a different auxotrophic locus and containing the amino acid synthesis gene to fill that auxotrophy as a marker for integration. The vectors are not suitable for Biobrick construction- they each contain one extraneous Biobrick site, and lack the standard cloning site. Thankfully, there is a standard method for cloning BBa parts onto the pRS30x vectors for yeast chromosomal integration: [2]

• Digest the BBa vector containing the finished construct and pRS30x vector of choice using the following enzymes:

*Alternatively, the final BBa construction ligation can be done as a double insertion onto a pRS30x vector. We did all of our pRS vector insertions this way. The three-way ligations work well consistently, and many variations (e.g. combination of promoters and coding regions) can be generated with relative ease:

• (Note: The orientation of the BioBrick part should be opposite that of the auxotropic gene once incorporated into the Sikorski vector.)

• Ligate the insert and vector using standard techniques and transform into E. coli
• Miniprep the vector from the transformed strain
• Linearize ~1 microgram of the vector per transformation using the appropriate enzyme according to the following table:

• Heat-kill and/or purify the digest
• Perform yeast transformation using a standard lithium acetate procedure. We have gotten good results from the following:

http://www.natureprotocols.com/2007/01/31/highefficiency_yeast_transform.php

[1] http://www.genetics.org/cgi/reprint/122/1/19.pdf
[2] http://openwetware.org/wiki/Silver:_BB_Strategy