Construction of a native promoter-containing transposon vector for the stable monitoring of the denitrifying bacterium Pseudomonas stutzeri LYS-86 by chromosomal-integrated gfp

Wei, Min; Kong, Zhuoyi; Zhong, Li; Qiu, Lequan; Li, Yansheng; Zhao, Lei; Li, Xuanzhen; Zhong, Wenbin

doi:10.1016/j.plasmid.2012.02.002

Cited by 3 publications

(2 citation statements)

References 13 publications

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“…It is generally accepted that the efficiency of regulatory elements (promoters) is higher than foreign promoters for chloroplast transformation (Fukuda et al 2008;Ruhlman et al 2010;Wei et al 2012). Prrn (Svab and Maliga 1993;Sidorov et al 1999), PpsbA (Li et al 2011), TpsbA (Svab and Maliga 1993;Sidorov et al 1999), and TrbcL (Doetsch et al 2001;Cui et al 2014) are commonly used endogenous regulators.…”

Section: Discussionmentioning

confidence: 99%

Enhanced green fluorescent protein (egfp) gene expression in Tetraselmis subcordiformis chloroplast with endogenous regulators

Cui

Zhao

Hou

et al. 2016

World J Microbiol Biotechnol

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On the basis of fundamental genetic transformation technologies, the goal of this study was to optimize Tetraselmis subcordiformis chloroplast transformation through the use of endogenous regulators. The genes rrn16S, rbcL, psbA, and psbC are commonly highly expressed in chloroplasts, and the regulators of these genes are often used in chloroplast transformation. For lack of a known chloroplast genome sequence, the genome-walking method was used here to obtain full sequences of T. subcordiformis endogenous regulators. The resulting regulators, including three promoters, two terminators, and a ribosome combination sequence, were inserted into the previously constructed plasmid pPSC-R, with the egfp gene included as a reporter gene, and five chloroplast expression vectors prepared. These vectors were successfully transformed into T. subcordiformis by particle bombardment and the efficiency of each vector tested by assessing EGFP fluorescence via microscopy. The results showed that these vectors exhibited higher efficiency than the former vector pPSC-G carrying exogenous regulators, and the vector pRFA with Prrn, psbA-5 0 RE, and TpsbA showed the highest efficiency. This research provides a set of effective endogenous regulators for T. subcordiformis and will facilitate future fundamental studies of this alga.

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Section: Discussionmentioning

confidence: 99%

Enhanced green fluorescent protein (egfp) gene expression in Tetraselmis subcordiformis chloroplast with endogenous regulators

Cui

Zhao

Hou

et al. 2016

World J Microbiol Biotechnol

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“…GFP emits bright visible fluorescence when expressed in a recombinant manner, and no substrate is required (4,5). The gfp gene has been expressed in many bacteria (6,7). Therefore, construction of a GFP-expressing strain of A. actinomycetemcomitans would enable the analysis of the dynamics of this species in experimental animals.…”

Section: Introductionmentioning

confidence: 99%

Transformation of <i>Aggregatibacter actinomycetemcomitans</i> with Green Fluorescent Protein

2016

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Green fluorescent protein (GFP) is one of the most useful biological markers for monitoring gene expression and the localization of particular cells. We constructed a GFPexpressing Aggregatibacter actinomycetemcomitans transformant by transposon insertion.This anaerobic, oral, gram-negative bacterium is thought to be the major causative agent in localized aggressive periodontitis and possesses the gene coding for leukotoxin (ltx). As this gene is stably expressed, the region extending from the ltx promoter to the initiation codon was amplified using PCR and temporarily cloned into a small Escherichia coli plasmid, pResKm. The DNA fragment containing the GFP gene(gfp)ORF was excised from the commercially available plasmid pAcGFP1-C1, purified by gel electrophoresis, and inserted downstream of the ltx initiation codon in the correct reading frame. This gene fusion was further cloned into the NotI site of the commercially available shuttle plasmid, pUTminiTn5, which contains the gene coding for the transposase, and then introduced into the chromosome of A. actinomycetemcomitans strain HK1651 by electroporation.Transformants were isolated by screening for the appropriate antibiotic resistance. FACS analysis revealed that the final A. actinomycetemcomitans-GFP integrant exhibited slightly more intense fluorescence than wild-type A. actinomycetemcomitans. These data demonstrate that transposon-mediated chromosomal integration is a useful approach for constructing mutant A. actinomycetemcomitans strains.

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Diversity and Abundance of Denitrifying Bacteria in the Sediment of a Eutrophic Estuary

Ming

Fan

Chen

et al. 2020

Geomicrobiology Journal

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Construction of a native promoter-containing transposon vector for the stable monitoring of the denitrifying bacterium Pseudomonas stutzeri LYS-86 by chromosomal-integrated gfp

Cited by 3 publications

References 13 publications

Enhanced green fluorescent protein (egfp) gene expression in Tetraselmis subcordiformis chloroplast with endogenous regulators

Enhanced green fluorescent protein (egfp) gene expression in Tetraselmis subcordiformis chloroplast with endogenous regulators

Transformation of <i>Aggregatibacter actinomycetemcomitans</i> with Green Fluorescent Protein

Diversity and Abundance of Denitrifying Bacteria in the Sediment of a Eutrophic Estuary

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