Inducible expression of heterologous genes targeted to a chromosomal platform in the cyanobacterium Synechococcus sp. PCC 7942

Abstract

High-level, inducible expression of heterologous genes in the cyanobacterium Synechococcus sp. strain PCC 7942 was obtained using the Escherichia coli trc promoter and lac/ repressor. The pet€ gene of Anabaena sp. strain PCC 7937 encoding plastocyanin precursor protein and the €. coli uidA gene encoding )?-glucuronidase were initially placed under the control of the trc promoter and lac/ 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 pTrclS 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 €. coli trc/lac/ system in Synechococcus was determined using Pglucuronidase as a reporter. The Anabaena PCC 7937 pet€ gene in Synechococcus was expressed to a high level upon induction with IPTG as shown by RNA and immunoblot analysis. The general usability of pTrclS as a cloning vector for inducible heterologous gene expression in Synechococcus was confirmed by the introduction of several more genes.

Cost-Effective and Uniform 13C- and 15N-Labeling of the 24-kDa N-Terminal Domain of the Escherichia coli Gyrase B by Overexpression in the Photoautotrophic Cyanobacterium Anabaena sp. PCC 7120

“…While some essential genes striction enzymes to excise the region coding for the 43-have already been identified (22,23, and references kDa N-terminal domain and replace it by that encoding therein), it has also been shown that it is possible to the 24-kDa domain. The resulting plasmid was named express heterologous proteins in this strain (24,25), pBL24K (Fig. 1A).…”

The Circadian Clock-Related Gene pex Regulates a Negative cis Element in the kaiA Promoter Region

“…The construct was recombined into a specific site of the genome, NSII, in which DNA insertion per se did not affect the fundamental cell activity or circadian rhythm. And at NSII, the inducible promoter trc used here had functioned in Synechococcus (6,9,17). To control the down-regulation of kaiA expression, we used the transcription-translation interference technique, using antisense transcription in Synechococcus (25).…”

“…Molecular genetic techniques, e.g., efficient transformation and gene targeting by precise homologous recombination, have been used for studying the cyanobacterial Synechococcus sp. strain PCC 7942 (3,6,24). By integrating molecular genetic properties and a monitoring system for the in vivo gene expression of bioluminescence, we previously isolated several types of clock mutants that exhibited altered bioluminescence rhythms, including short-or long-period rhythms and even arrhythmias (16).…”

Expression of the Copper Metallothionein CUPI from Saccharomyces cerevisiae in the Cyanobacterium Synechococcus R2-PIM8(smtA)

“…strain PCC 7942 R2-PIM8 (smtA) [7] to produce an organism with enhanced toxic metal sequestration capability. Metallothioneins are low-molecular-weight, cysteine-rich proteins that chelate certain metals as mercaptide complexes [14].…”

“…All plasmids were propagated in E. coli DH5␣. The pTrcIK cloning vector is described in [7]. The plasmid pJW6 [6] was the source for the Saccharomyces cerevisiae CUPI gene.…”