In order to develop useful plasmid vectors for Zymomonas cells, attempts were made to isolate some natural plasmids from Z.mobilis ATCC10988. Among a few plasmid isolated, a small plasmid having 2.7Kb in size was chosen and designated as pZM2.
A restriction map of pZM2 was made and found to contain one cleavage site for AvaI, BamHI, BglII and two for HindIII.
By introducing the replication origin of pZM2 into pBR325, hybrid plasmid vector was constructed and designated as pHZ22.
This vector contained chloramphenicol resistance gene as a genetic marker and proved to be stably maintained in Z.mobilis ZM4. Another trial was made to construct a better plasmid containing tetracycline resistance gene. This gene was isolated from RP4 and introduced into pHZ22 to make a new vector called pHZT224. Through a series of experiments, it was evident that these plasmid vector containing selectable markers of chloramphenicol and tetracycline resistance was a shuttle vector which was functional in Z.mobilis as well as in E.coli.