Inducible expression of heterologous genes targeted to a chromosomal platform in the cyanobacterium Synechococcus sp. PCC 7942.
High-level, inducible expression of heterologous genes in the cyanobacterium Synechococcus sp. strain PCC 7942 was obtained using the Escherichia coli trc promoter and lacI repressor. The petE gene of Anabaena sp. strain PCC 7937 encoding plastocyanin precursor protein and the E. coli uidA gene encoding beta-glucuronidase were initially placed under the control of the trc promoter and lacI repressor by cloning into the E. coli pTrc99C expression vector and were introduced into the chromosomal platform for integration in metF (PIM) of the Synechococcus R2-PIM9 recipient strain. These pTrc99C-derived constructs often gave rise to transformants that did not contain a complete insert gene, probably because of gene conversion events. Selection of the desired Synechococcus R2-PIM9 transformants was vastly improved using the new pTrcIS vector that contains the aadA gene encoding streptomycin resistance as an extra antibiotic resistance marker. The influence of IPTG concentration and induction time on gene expression with the E. coli trc/lacI system in Synechococcus was determined using beta-glucuronidase as a reporter. The Anabaena PCC 7937 petE gene in Synechococcus was expressed to a high level upon induction with IPTG as shown by RNA and immunoblot analysis. The general usability of pTrcIS as a cloning vector for inducible heterologous gene expression in Synechococcus was confirmed by the introduction of several more genes.
Geerts
D
D
Department of Molecular Cell Biology, University of Utrecht, The Netherlands.
Bovy
A
A
de Vrieze
G
G
Borrias
M
M
Weisbeek
P
P
D000595:Amino Acid Sequence; D017033:Anabaena / Q000235:genetics; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D017930:Genes, Reporter; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D014170:Transformation, Genetic
D004269:DNA, Bacterial