RESUMO
An expression vector, pFC1, has been constructed based on the promiscuous plasmid RSF1010, which provides autonomous replication in several cyanobacteria of the genera Synechocystis and Synechococcus [Mermet-Bouvier et al., Curr Microbiol 26:323-327]. pFC1 harbors the lambda cI857 repressor-encoding gene and pR promoter, followed by the lambda cro ribosome-binding site and ATG translation initiation codon. The latter is located within the unique NdeI restriction site (CATATG) of pFC1 and can be exposed after cleavage with this enzyme for in-frame fusion with the protein-coding sequence to be expressed. The Escherichia coli lacZ reporter gene cloned in pFC1 appeared to be highly expressed in heat-induced E. coli or cyanobacterial cells. In every case, beta-galactosidase amounted to at least 10% of soluble proteins.
Assuntos
Cianobactérias/genética , Vetores Genéticos , Plasmídeos/genética , Sequência de Bases , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica , Dados de Sequência Molecular , Especificidade da Espécie , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
A promoter-probe vector, pSB2A, based on the plasmid RSF1010 and the promoterless chloramphenicol acetyl transferase (cat) reporter gene, has been constructed. pSB2A appeared to be most efficiently transferred by conjugation to the widely used cyanobacteria Synechocystis strains PCC6803 (S.6803) and PCC6714 (S.6714) and Synechococcus strains PCC7942 (S.7942) and PCC6301 (S.6301), where it replicates stably even though it contains no cyanobacterial DNA. Using pSB2A we found that (1) a light-regulated promoter from S.6803 remains controlled by light intensity in S.7942 while it is silent in Escherichia coli, and (2) the E. coli tac promoter behaves as a strong and light-independent promoter in the four cyanobacterial hosts tested.