CONSTRUCTION OF MULTIPURPOSE GENE CARTRIDGES BASED ON A NOVEL SYNTHETIC PROMOTER FOR HIGH-LEVEL GENE-EXPRESSION IN GRAM-NEGATIVE BACTERIA

A series of gene cartridges containing a novel synthetic promoter (P-s yn) was constructed. The P-syn sequence is based on the consensus of a number of naturally occurring promoters and displays strong activity in Escherichia coli and Rhizobium leguminosarum. In a direct compariso n, P-syn proved to be about twice as strong as the tac promoter in E. coli, while the difference in Rhizobium was about tenfold. A small P-s yn cartridge was constructed by adding a Shine-Dalgarno sequence, an A TG codon, and a removable lac operator, whose excision can convert the regulated cartridge into a constitutively expressed unit. A second ca ssette was obtained by the addition of a lacl(q) gene in order to prov ide autonomous regulation also in hosts lacking lacI functions, such a s R. leguminosarum. A promoterless lacZ gene was inserted to monitor t he activity. This gene can be either replaced with genes of interest, or used for gene fusions by means of conveniently positioned restricti on sites. A third cassette was generated by adding a mercury-resistanc e determinant as a selectable marker, suitable for monitoring tagged b acteria released into environments. In such cases, where a non-antibio tic-resistant marker is preferrable, the use of mercury chloride adds the advantage of inhibiting fungal growth when plating soil suspension s. The presence of he second marker, lacZ driven by the strong P-syn, facilitates the selection. Furthermore, the P-syn fragment can be used as a specific probe for the detection of released bacteria engineered with any of the above constructs.