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GFP expression at the RNA level in ABRG5-3 transconjugants. a RT-PCR. The ABRG5-3 transconjugants harboring pGFP500 (+AU) or pGFP461c (−AU) were grown under continuous white light illumination (35 μE m−2 s−1) in the BG11 medium for 12 days. The cells were harvested, total RNA (0.1 μg) without genomic DNA was prepared from the cells, and then cDNA (0.1 μg) was synthesized from the template RNA with the GFP-specific primer GFP-RT and reverse transcriptase. The RNA and cDNA were subjected to RT-PCR with the specific primers GFP-F and GFP-RT. The position of 0.72 kb for gfp is shown at the left. b QRT-PCR. The respective RNAs and cDNA were subjected to QRT-PCR with the GFP-specific primers GFP-F and GFP-QRT. The relative positions of each primer within the gfp gene are shown at the top. Details for the PCR were described previously (Asayama et al. 2004). Relative expression levels for transcripts were calculated with a 100 % value referring to that of cDNA in the GFP461c strain. Relative average values (in percent) are shown as bars with error values (standard deviation, SD) obtained from independent triplicate experiments
The accumulation of GFP transcripts in the transconjugants of
ABRG5-3 was examined by PCR (Fig. 9).
Asayama, MunehikoJournal: Applied Microbiology and Biotechnology
Issue 3DOI: 10.1007/s00253-012-3989-0Published: 2012-07-12Institution(s):
New cyanobacterial expression vectors, possessing an origin of replication that functions in a broad range of Gram-negative bacteria, were constructed. To inspect the shuttle vectors, the gene gfp was cloned downstream from the expression control element (ECE) originating from the regulatory region of the Microcystis aeruginosa gene psbA2 (for photosystem II D1 protein), and the vectors were introduced into three kinds of cyanobacteria (Synechocystis sp. PCC 6803, Synechococcus elongatus PCC 7942, and Limnothrix/Pseudanabaena sp. ABRG5-3) by conjugation. Multiple copy numbers of the expression vectors (in the range of 14–25 copies per cell) and a high expression of green fluorescent protein (GFP) at the RNA/protein level were observed in the cyanobacterial transconjugants. Importantly, GFP was observed in a supernatant from the autolysed transconjugants of ABRG5-3 and easily collected from the supernatant without centrifugation and/or further cell lysis. These results indicate the vectors together with the recombinant cells to be useful for overproducing and recovering target gene products from cyanobacteria.
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