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. 2019 Dec 5;13(2):339–349. doi: 10.1111/1751-7915.13516

Figure 1.

Figure 1

RT‐PCR of viable H. thermocellum transformants, containing the pThermoCas9i plasmid. A. Schematic illustration of plasmid pThermoCas9i_NT (Mougiakos et al., 2017). Thermodcas9 gene under control of the B. smithii xylL promoter; sgRNA‐expressing module under control of the B. coagulans pta promoter; and pNW33n backbone. B, C and D. Non‐targeting 1 and 2, independent pThermoCas9i_NT (Mougiakos et al., 2017) transformants of H. thermocellum (lanes 2 and 3) showing the RT‐PCR products of 124 bp (B) and 282 bp (C) from thermodcas9 cDNA and the absence of the RT‐PCR product of 169 bp (D) from the sgRNA; WT, RT‐PCR with H. thermocellum DSM 1313 wild‐type cDNA; B. smithii, RT‐PCR with B. smithii harbouring pThermoCas9i_NT (Mougiakos et al., 2017) cDNA. E) MQ, RT‐PCR with Milli‐Q water (lane 1); WT, RT‐PCR with H. thermocellum DSM 1313 wild‐type DNA (lane 2); WT, RT‐PCR with H. thermocellum DSM 1313 wild‐type RNA (lane 3); Non‐targeting 1 and 2, RT‐PCR with H. thermocellum transformants 1 and 2 RNA, respectively, (lane 4 and 5); and B. smithii, RT‐PCR with B. smithii harbouring pThermoCas9i_NT (Mougiakos et al., 2017) cDNA (lane 6).