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Fig. 2 | Biotechnology for Biofuels

Fig. 2

From: Determination of the native features of the exoglucanase Cel48S from Clostridium thermocellum

Fig. 2

Knock–in of a sequence encoding 12 successive histidine residues (His12) and a TAA stop codon in the chromosome of C. thermocellumpyrF. 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

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