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-syn) 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 comparison, 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-syn cartridge was constructed by adding a Shine-Dalgarno sequence, an ATG codon, and a removable lac operator, whose excision can convert the regulated cartridge into a constitutively expressed unit. A second cassette was obtained by the addition of a lacl(q) gene in order to provide autonomous regulation also in hosts lacking lacI functions, such as R. leguminosarum. A promoterless lacZ gene was inserted to monitor the activity. This gene can be either replaced with genes of interest, or used for gene fusions by means of conveniently positioned restriction sites. A third cassette was generated by adding a mercury-resistance determinant as a selectable marker, suitable for monitoring tagged bacteria released into environments. In such cases, where a non-antibiotic-resistant marker is preferrable, the use of mercury chloride adds the advantage of inhibiting fungal growth when plating soil suspensions. 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.