Attempted Construction of a soxR::PsoxS::lacZ Recombinant pBR322 Plasmid as a Sensitive Reporter of Internal Reactive Oxygen Species in Escherichia coli DH5α

This study attempted to engineer an internal reactive oxygen species probe using lacZ expression driven by the soxS promoter in response to intracellular superoxide presence being sensed by SoxR. Using polymerase chain reaction amplification, soxR fused to the promoter of soxS (P ) and lacZ amplicons were obtained. Double soxS restriction digests and sticky-end ligations were then performed in an attempt to clone soxR:: P::lacZ into the soxS pBR322 plasmid vector. Following ligation, transformation of competent Escherichia coli DH5α by calcium chloride-heatshock transformation was performed, and resulting transformants were screened for the soxR::P::lacZ::pBR322 construct. Even though the amplicons were made, a successful ligation was not achieved. soxS The lack of success in obtaining the desired construct prevented further examination of the potential of the proposed reactive oxygen species probe.