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We applied a point mutation to the Pcold in order to be easy to clone it. We changed the 178th base C to A. An article which we referred describes as stated below.
“We found occasionally that C–A base substitution at nucleotide number 532 in the 5’-UTR region (nucleotide number is according to GenBank Accession No. M30139) diminished this growth inhibition effect (data not shown), allowing easy cloning of the genes of interest into MCS. We do not know why this mutation diminished growth inhibition effect but the mutation did not affect productivity of proteins (data not shown).”
Nakashima,N and Tamura,T. 2004. Cell-free protein synthesis using cell extract of Pseudomonas fluorescens and CspA promoter. Biochemical and Biophysical Research Communications 319 (2004) 672