Unusual observations during construction of a new cloning vector providing Ion gene expression in Escherichia coli

The plasmid pJMC40 containing the Ion gene was transformed into E. coil HB101 leading to clones with either pJMC40 or a large plasmid designated pJMC40ins, pJMC40ins has a 1.3 kb D N A insert outside of the Ion operon. Attempts to reduce the size of pJMC40 through circularization of a 6.7 kb EcoRI fragment containing the Ion operon and larger parts of pBR322 failed. Circularization of an 8.0 kb EcoRI fragment of pJMC40ins, consisting of the 6.7 kb fragment and the 1.3 kb insert, produced a new plasmid which is useful as a small vector with a functional Ion operon. The Ion gene was cloned in vitro into the vector pUC18 setting it under the control of the lac promotor. Different attempts to transform this construct into E. coli failed. We suggest that the expression level of Ion is very crucial for the viability of E. coli cells. Changes in regulation of Ion expression expected to elevate La activity are lethal for E. coli.