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. Author manuscript; available in PMC: 2019 Jul 20.
Published in final edited form as: ACS Synth Biol. 2018 Jul 10;7(7):1722–1729. doi: 10.1021/acssynbio.8b00195

Figure 1. Engineering recombinant p2.

Figure 1.

(A) Top: AR-Y292 strain harboring p1 and p2, replicated by self-encoded TP-DNAP1 and TP-DNAP2, respectively. Middle: Intermediate strain derived from AR-Y292 by integration of mKate2, URA3, and leu2* over ORF1 of p2 and expression of Cas9 targeting ORF1 of p2, which is missing from the recombinant p2-delORF1-mUL*. Bottom: Complete curing of the parental p2 plasmid induced by Cas9 results in GA-Y021, which contains only p1 and recombinant p2-delORF1-mUL*. GA-Y021 is used as the base strain for subsequent measurements of p2 mutation rate. (B) Gel analysis (0.9% agarose) verifying integration of mKate2, URA3, leu2* and loss of parental p2 after Cas9 treatment. Lane 1: DNA extracted from AR-Y292, containing linear plasmids p1 and p2. Lane 2: DNA extracted from GA-Y021, confirming loss of p2 and its replacement by p2-delORF1-mUL* at high copy.