Team:BrownTwo/Protocols/bbyeastchrom

From 2008.igem.org

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:

Sikorski vector (marker)	insert & vector digest
pRS303 (HIS3)	EcoRI, SpeI
pRS304* (TRP1)	EcoRI, SpeI
pRS305 (LEU2)	XbaI, PstI
pRS306 (URA3)	EcoRI, SpeI

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:

Sikorski vector	vector digest	forward part digest	back part digest
pRS303 (HIS3)	EcoRI, Not I	EcoRI, SpeI	XbaI, NotI
pRS304* (TRP1)	EcoRI, Not I	EcoRI, SpeI	XbaI, NotI
pRS305 (LEU2)	NotI, SpeI	NotI, PstI	XbaI, PstI
pRS306 (URA3)	EcoRI, Not I	EcoRI, SpeI	XbaI, NotI

(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:

Vector	Marker	Linearization Enzyme
pRS303	HIS3	PstI
pRS304*	TRP1	PstI
pRS305	LEU2	BstEII
pRS306	URA3	PstI

Heat-kill and/or purify the digest
Transform the linear construct into a haploid strain with the correct auxotrophies (His, Trp, Leu, Ura- we used W303) 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

Select for transformants by plating the transformation on a synthetic defined (SD) plate with amino acid dropouts for whatever gene is encoded on the vector you transformed. Double integrations (integrating two constructs at ones) worked very reliably for us, and triple integrations worked about half the time. If you need to integrate more than four constructs, you can transform up to three into each haploid mating type (a and alpha), then mate them to yield a diploid strain with all of your constructs. Use complementary selection markers (e.g. His, Trp, Leu in type a and Trp, Leu, Ura in type alpha) so you can plate the mixed cells on SD-His-Trp-Leu-Ura and select for cells that have mated, and thus have all of your constructs in their genome.

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