McClean: Working with the LoxP/Cre System in S. cerevisiae
The loxP/Cre recombinase system is an extremely powerful tool for genetic manipulation, allowing for conditional and site-specific integrations, excisions, and inversions, either in vivo or in vitro.
The loxP element is a 34 bp element, consisting of an 8bp region that confers directionality, flanked by 13 bp palindromic regions. The orientation of pairs of loxP sites determines the recombination outcome upon exposure to Cre recombinase. DNA in between loxP sites in opposing orientation is inverted while that in between sites facing the same direction is excised as a DNA circle, leaving a single loxP site, or "scar." Further, a pair of DNA molecules, at least one circular, each with a single loxP site, can be fused.
Using loxP/Cre in vivo with Yeast
Better Method- Using Phleomycin
- Transform pMM296 (pSH65 Carter and Delneri, 2010) into the strain of interest, plate on YPD, and replica plate onto YPD + 10 μg/mL Phleomycin the next day. The manufacturer's instructions are found here: Invivogen Product Information, Phleomycin.
Old Method-Using Zeocin (Yeast are poorly sensitive to Zeocin)
- A common use for the system in yeast is excising a selectable marker previously integrated into the genome. In our instance, we would transform pMM296 (pSH65 Carter and Delneri, 2010) into the strain of interest, plate on YPD, and replica plate onto YPD + 150 μg/mL Zeocin the next day (If your marker is counterselective, you can alternatively select for it's loss instead.) After you confirm the excision, lose pMM296 with the Plasmid Loss Assay.
Using loxP/Cre in vitro
Alternatively, using commercially available Cre recombinase (NEB #M0298S, among others,) you can excise in vitro. Follow the NEB protocol (https://www.neb.com/protocols/2014/02/12/protocol-for-cre-recombinase-m0298 and/or pg 118 of the 2013-2014 NEB catalog.) Be sure to use the pLox2+ control. Note that the Cre-mediated recombination is an equilibrium reaction, resulting in about 20 to 30% recombination. As a result, you should either gel purify your desired product, or, if you can't get good enough resolution to cut the band out and your desired product is a plasmid that will replicate, transform the whole reaction mixture and screen with colony PCR.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
- Michael T. Patel 18:36, 24 July 2014 (EDT):
or instead, discuss this protocol.