Computational protocol: The Cytoplasmic and Periplasmic Expression Levels and Folding of Organophosphorus Hydrolase Enzyme in Escherichia coli

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Protocol publication

[…] The sequence encoding the OPH gene from Pseudomonas diminuta (Expasy accession No.P0A434) was designed and optimized for E. coli by the JCat software (), Gen Script´s (http://www.genscript.com/cgi-bin/tools/rare_codon_analysis) and Mfold () software); then, it was synthesized (Biomatik Corp., Canada) into both pET21a and pET26b plasmids. The synthesized sequence, including a 1008-bp fragment with an additional adenine base in the pET26b plasmid, was approved using double-digestion method by 5´ BamHI and HindIII 3´enzymes. E. coli BL21 was transformed using a MicroPulser (Bio-Rad) at 1200V in a 0.1 cm electroporation cuvette. The preparation of electrocompetent cells and electroporation were carried out as described in Bio-Rad MicroPulser electroporation apparatus operating instructions and applications guide (Bio-Rad, #165 - 2100). […]

Pipeline specifications

Software tools Jcat, Mfold
Databases ExPASy
Application Synthetic biology
Organisms Escherichia coli
Chemicals Organophosphates