Figure 3
From: Genetic engineering of Pyrococcus furiosus to use chitin as a carbon source
Cloning strategy for the mutant MUR23Pf. Plasmid pMUR47 was constructed by overlapping PCR. It contained the chitinase sequence with the deleted nucleotide at position 1006 with corresponding upstream and downstream regions for homologous recombination together with the resistance cassette consisting of TK0914 for selection with the antibiotics simvastatin and PF1950 for the counter selection with 6-methylpurine. After transformation with linearized pMUR47 the intermediate mutant MUR23Pf_i was isolated and characterized. The negative selection with 6-methylpurine resulted in strain MUR23Pf with the redesigned chitinase.