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Figure 1 | Biotechnology for Biofuels

Figure 1

From: Overcoming restriction as a barrier to DNA transformation in Caldicellulosiruptor species results in efficient marker replacement

Figure 1

Strategy for construction and PCR analysis of the cbeI (Cbes2438) deletion in JWCB005. (A) A diagram of the cbeI genome region is shown with the cbeI knock-out plasmid having ~ 0.5 kb regions from each up- and downstream of cbeI for homologous recombination and also containing the pyrF cassette [13] for selection of transformants. Homologous recombination can occur at the upstream or downstream cbeI flanking regions, integrating the plasmid into the genome and generating a strain that is a uracil prototroph. Counter-selection with 5-fluoroorotic acid (5-FOA) selects for loss of the plasmid and deletion of the cbeI gene. Bent arrows depict primers used for verification of the cbeI deletion. (B) Gel depicting PCR products amplified from the cbeI genome region in JWCB018 (ΔpyrFAcbeI) compared to the parental strain JWCB005 (ΔpyrFA), amplified by primers (DC277 and DC239). Lane 1: JWCB005; lane 2: JWCB018; M: 1 kb DNA ladder (NEB).

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