Fig. 2

Knock–in of a sequence encoding 12 successive histidine residues (His₁₂) and a TAA stop codon in the chromosome of C. thermocellum ∆pyrF. a Schematic representation of the workflow of the seamless genome-editing system using the plasmid pHK–HR–His₁₂TAA. The first round of recombination used the combined selection of FUDR toward Tdk and uracil auxotrophic medium toward PyrF after transformation of the plasmid into ∆pyrF strain. The second round of recombination occurred in the presence of the FOA selection stress. The pyrF cassette was removed. Compared with the parent strain ∆pyrF, the target mutant ∆pyrF::Cel48S_CD-His₁₂ produced the Cel48S_CD protein containing a His₁₂-tag (GH48-His₁₂), instead of the type I dockerin (GH48-Doc). b Diagnostic PCR investigation of the mutant strain after each recombination step. M, DNA marker. c SDS-PAGE analysis of the extracellular proteins of ∆pyrF (Lane 1) and ∆pyrF::Cel48S_CD-His₁₂ (Lane 2) strains. The supernatants were tenfold condensed (10–1 mL) by ultrafiltration (10.0 kDa cutoff) (Merck Millipore, Billerica), and approximately 5 μg of the protein samples was loaded in each lane. The bands corresponding to known cellulosomal proteins are identified to the left of the Coomassie blue-stained gel. The extracellular proteins of ∆pyrF::Cel48S_CD-His₁₂ was purified by Ni²⁺ affinity chromatography and subsequently, by gel filtration. Lanes 3 and 4 show the SDS-PAGE analysis of the eluted fractions, respectively. M, protein standards