pET-INP-cellulase

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	The ligation mixture was transformed into E. coli BL21DE3, and the transformants were screened on LB agar plates supplemented with ampicillin (100 μg/mL). The multiple nucleotide sequence analysis of the recombinant was confirmed with Clustal Omega software, and the amino acid sequence alignment was performed using BLASTP at the NCBI.

Protocol tips
The ligation mixture was transformed into E. coli BL21DE3, and the transformants were screened on LB agar plates supplemented with ampicillin (100 μg/mL). The multiple nucleotide sequence analysis of the recombinant was confirmed with Clustal Omega software, and the amino acid sequence alignment was performed using BLASTP at the NCBI.

Publication protocol

The chromosomal DNA-containing cellulase sequence of B. licheniformis ATCC 14580 was prepared and isolated with the JetFlex genomic DNA purification kit. The putative cellulase gene (1683 bp) from the genomic sequences was amplified via polymerase chain reaction (PCR) with two designed primers: forward primer 5′-GCTTGCGGCCGCTTGAGAGAAAAATGG-3′ and reverse primer 5′-GAGTGCGGCCGCAGGXGTGATTTTCACC-3′ (the recognition enzyme restriction site of Not I is underlined). Polymerase chain reactions were carried out in a Perkin-Elmer thermal cycler (GeneAmp PCR System 9700, Norwalk, CT, USA) for 30 cycles. Each cycle was composed of a denaturation stage at 94 °C for 45 s, an annealing stage at 56 °C for 2 min, and extension stage at 72 °C for 2 min. The amplified PCR fragments were purified via the PCR clean-up kit, digested by the Not I restriction enzyme, and then ligated into the corresponding restriction site of the digested vector. The ligation mixture was transformed into E. coli BL21DE3, and the transformants were screened on LB agar plates supplemented with ampicillin (100 μg/mL). The multiple nucleotide sequence analysis of the recombinant was confirmed with Clustal Omega software, and the amino acid sequence alignment was performed using BLASTP at the NCBI.

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