Knock-in of gene caBglA in the chromosome of C. thermocellum ∆pyrF.
a Schematic illustrating the work flow of the seamless genome editing system using plasmid pHK-HR-CaBglA. Three screening steps including two rounds of recombination are involved. The first step consists of the transformation of plasmid into ∆pyrF strain, and the selection on Tm. The second step employs the combined selection of FUDR and uracil auxotrophic MJ medium to promote the integration of the “PyrF-HR-short-CaBglA” fragment onto the chromosome and the elimination of the transformed plasmid. The PyrF function of the host cell was restored in this step. In the third step, the FOA selection stress promotes the removal of pyrF cassette through the second round of recombination. b Diagnostic PCR investigation of the obtained recombinant strain after each selection step. The target strain ∆pyrF::CaBglA shows a PCR product of 4.7 kb, indicating the successful knock-in of caBglA gene in the chromosome. M DNA marker