A plasmid facilitatingin vitroconstruction of phoA gene fusions inEscherichia coli

The topology of membrane proteins is technically difficult to determine. Genetic approaches have been described which help for such an analysis (5). In E. coli, alkaline phosphatase is enzyinatically active only when it is exported out of the cytoplasm of the cell (1) and the use of gene fusions with phoA, the gene for alkaline phosphatase, revealed the more generally applicable of these genetic methods (4,5). phoA fusions can be obtained in vivo, by random transposition of the transposon TnphoA (3), or in vitro, using plasmids in which the phoA sequence is preceded by a PstI site (2) . We present here a new plasmid which will facilitate the in vitro construction of phoA fusions.