Modification of RSF1010-Based Broad-Host-Range Plasmids for Improved Conjugation and Cyanobacterial Bioprospecting

Bishé, Bryan; Taton, Arnaud; Golden, James W.

doi:10.1016/j.isci.2019.09.002

Cited by 36 publications

(29 citation statements)

References 39 publications

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“…Such strategy could perhaps be used to promote the positive selection of RSF1010-derived replicative plasmids in the absence of antibiotics in Synechocystis [ 68 ], and characterized in combination with potentially toxic expression systems that counteract the effect by inducing plasmid instability. The preparative use of these plasmids can be further improved by the inactivation of the conjugation-associated mobA system that enhances extraction yields of intact plasmid dsDNA and thus the in vitro cloning efficiency [ 69 , 70 ], whenever natural transformation is used for generating the strains.…”

Section: Discussionmentioning

confidence: 99%

Comparison of alternative integration sites in the chromosome and the native plasmids of the cyanobacterium Synechocystis sp. PCC 6803 in respect to expression efficiency and copy number

et al. 2021

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Background Synechocystis sp. PCC 6803 provides a well-established reference point to cyanobacterial metabolic engineering as part of basic photosynthesis research, as well as in the development of next-generation biotechnological production systems. This study focused on expanding the current knowledge on genomic integration of expression constructs in Synechocystis, targeting a range of novel sites in the chromosome and in the native plasmids, together with established loci used in literature. The key objective was to obtain quantitative information on site-specific expression in reference to replicon copy numbers, which has been speculated but never compared side by side in this host. Results An optimized sYFP2 expression cassette was successfully integrated in two novel sites in Synechocystis chromosome (slr0944; sll0058) and in all four endogenous megaplasmids (pSYSM/slr5037-slr5038; pSYSX/slr6037; pSYSA/slr7023; pSYSG/slr8030) that have not been previously evaluated for the purpose. Fluorescent analysis of the segregated strains revealed that the expression levels between the megaplasmids and chromosomal constructs were very similar, and reinforced the view that highest expression in Synechocystis can be obtained using RSF1010-derived replicative vectors or the native small plasmid pCA2.4 evaluated in comparison. Parallel replicon copy number analysis by RT-qPCR showed that the expression from the alternative loci is largely determined by the gene dosage in Synechocystis, thereby confirming the dependence formerly proposed based on literature. Conclusions This study brings together nine different integrative loci in the genome of Synechocystis to demonstrate quantitative differences between target sites in the chromosome, the native plasmids, and a RSF1010-based replicative expression vector. To date, this is the most comprehensive comparison of alternative integrative sites in Synechocystis, and provides the first direct reference between expression efficiency and replicon gene dosage in the context. In the light of existing literature, the findings support the view that the small native plasmids can be notably more difficult to target than the chromosome or the megaplasmids, and that the RSF1010-derived vectors may be surprisingly well maintained under non-selective culture conditions in this cyanobacterial host. Altogether, the work broadens our views on genomic integration and the rational use of different integrative loci versus replicative plasmids, when aiming at expressing heterologous genes in Synechocystis.

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Section: Discussionmentioning

confidence: 99%

Comparison of alternative integration sites in the chromosome and the native plasmids of the cyanobacterium Synechocystis sp. PCC 6803 in respect to expression efficiency and copy number

et al. 2021

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“…The pAM5505 plasmid (Addgene #132664) constructed by Elhai et al. is a helper plasmid for conjugal transfer of DNA ( 17 ). It was developed for genetically engineering cyanobacteria.…”

Section: Resultsmentioning

confidence: 99%

pLannotate: engineered plasmid annotation

Barrick

2021

Nucleic Acids Research

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Engineered plasmids are widely used in the biological sciences. Since many plasmids contain DNA sequences that have been reused and remixed by researchers for decades, annotation of their functional elements is often incomplete. Missing information about the presence, location, or precise identity of a plasmid feature can lead to unintended consequences or failed experiments. Many engineered plasmids contain sequences—such as recombinant DNA from all domains of life, wholly synthetic DNA sequences, and engineered gene expression elements—that are not predicted by microbial genome annotation pipelines. Existing plasmid annotation tools have limited feature libraries and do not detect incomplete fragments of features that are present in many plasmids for historical reasons and may impact their newly designed functions. We created the open source pLannotate web server so users can quickly and comprehensively annotate plasmid features. pLannotate is powered by large databases of genetic parts and proteins. It employs a filtering algorithm to display only the most relevant feature matches and also reports feature fragments. Finally, pLannotate displays a graphical map of the annotated plasmid, explains the provenance of each feature prediction, and allows results to be downloaded in a variety of formats. The webserver for pLannotate is accessible at: http://plannotate.barricklab.org/

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“…The pDUOTK1-L1 vector contains the broad host range replicative origin RSF1010, which has been shown to be functional in a wide diversity of prokaryotic species, including cyanobacteria from all five subsections ( Mermet-Bouvier et al, 1993 ; Stucken et al, 2012 ; Bishé et al, 2019 ). Thus, pDUOTK1-L1 could help to make terminator characterization more accessible, as promising new strains are discovered ( Włodarczyk et al, 2019 ; Jaiswal et al, 2020 ; Nies et al, 2020 ).…”

Section: Discussionmentioning

confidence: 99%

Evaluation and Comparison of the Efficiency of Transcription Terminators in Different Cyanobacterial Species

Gale

McCormick

2021

Front. Microbiol.

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Cyanobacteria utilize sunlight to convert carbon dioxide into a wide variety of secondary metabolites and show great potential for green biotechnology applications. Although cyanobacterial synthetic biology is less mature than for other heterotrophic model organisms, there are now a range of molecular tools available to modulate and control gene expression. One area of gene regulation that still lags behind other model organisms is the modulation of gene transcription, particularly transcription termination. A vast number of intrinsic transcription terminators are now available in heterotrophs, but only a small number have been investigated in cyanobacteria. As artificial gene expression systems become larger and more complex, with short stretches of DNA harboring strong promoters and multiple gene expression cassettes, the need to stop transcription efficiently and insulate downstream regions from unwanted interference is becoming more important. In this study, we adapted a dual reporter tool for use with the CyanoGate MoClo Assembly system that can quantify and compare the efficiency of terminator sequences within and between different species. We characterized 34 intrinsic terminators in Escherichia coli, Synechocystis sp. PCC 6803, and Synechococcus elongatus UTEX 2973 and observed significant differences in termination efficiencies. However, we also identified five terminators with termination efficiencies of >96% in all three species, indicating that some terminators can behave consistently in both heterotrophic species and cyanobacteria.

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Modification of RSF1010-Based Broad-Host-Range Plasmids for Improved Conjugation and Cyanobacterial Bioprospecting

Cited by 36 publications

References 39 publications

Comparison of alternative integration sites in the chromosome and the native plasmids of the cyanobacterium Synechocystis sp. PCC 6803 in respect to expression efficiency and copy number

Comparison of alternative integration sites in the chromosome and the native plasmids of the cyanobacterium Synechocystis sp. PCC 6803 in respect to expression efficiency and copy number

pLannotate: engineered plasmid annotation

Evaluation and Comparison of the Efficiency of Transcription Terminators in Different Cyanobacterial Species

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