Overview
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 (Carter and Delneri, 2010)
- 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.
- Inoculate Phleomycin-resistant colonies in YP+raffinose and let it grow overnight to saturation.
- Harvest cells by centrifuging. Wash the pellets with water and resuspend the pellets in 10mL YP+galactose at OD_600 = 0.3 and incubated at 30 C for 2-4 h.
- Plate culture (preferably by serial dilutions) on to YPD plates and incubated for 1-2 days.
- Replica-plate onto SC+5’FOA and YPD+phleomycin. The desired colonies are those that only grow in SC+5’FOA and not YPD+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.