New Orange Ligand-Dependent Fluorescent Reporter for Anaerobic Imaging

Chia, Hannah E; Koebke, Karl J.; Rangarajan, Aathmaja Anandhi; Koropatkin, Nicole M.; Marsh, E. Neil G.; Biteen, Julie S.

doi:10.1021/acschembio.1c00391

Cited by 11 publications

(9 citation statements)

References 34 publications

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“…Successful application of DE-FACS to obtain P. faecium is therefore currently limited by the lack of highly-fluorescent dyes that work under fully anaerobic conditions. Recently, there has been promising work in creating anaerobic fluorescent reporter proteins [ 68 , 69 ], but microbial physiologists would greatly benefit from the creation of anaerobic fluorescent dyes for non-genetically-tractable systems. In addition to lack of strong fluorescent dye choices, some strict anaerobes, including possibly P. faecium , would likely not survive transient oxygen exposure during sorting on a commercial FACS instrument.…”

Section: Discussionmentioning

confidence: 99%

Double emulsions as a high-throughput enrichment and isolation platform for slower-growing microbes

McCully

Yao

Brower

et al. 2023

ISME Communications

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Our understanding of in situ microbial physiology is primarily based on physiological characterization of fast-growing and readily-isolatable microbes. Microbial enrichments to obtain novel isolates with slower growth rates or physiologies adapted to low nutrient environments are plagued by intrinsic biases for fastest-growing species when using standard laboratory isolation protocols. New cultivation tools to minimize these biases and enrich for less well-studied taxa are needed. In this study, we developed a high-throughput bacterial enrichment platform based on single cell encapsulation and growth within double emulsions (GrowMiDE). We showed that GrowMiDE can cultivate many different microorganisms and enrich for underrepresented taxa that are never observed in traditional batch enrichments. For example, preventing dominance of the enrichment by fast-growing microbes due to nutrient privatization within the double emulsion droplets allowed cultivation of slower-growing Negativicutes and Methanobacteria from stool samples in rich media enrichment cultures. In competition experiments between growth rate and growth yield specialist strains, GrowMiDE enrichments prevented competition for shared nutrient pools and enriched for slower-growing but more efficient strains. Finally, we demonstrated the compatibility of GrowMiDE with commercial fluorescence-activated cell sorting (FACS) to obtain isolates from GrowMiDE enrichments. Together, GrowMiDE + DE-FACS is a promising new high-throughput enrichment platform that can be easily applied to diverse microbial enrichments or screens.

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Section: Discussionmentioning

confidence: 99%

Double emulsions as a high-throughput enrichment and isolation platform for slower-growing microbes

McCully

Yao

Brower

et al. 2023

ISME Communications

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“…ljungdahlii in synthetic co-cultures using FAST and the HaloTag as reporters. , Still, the HaloTag and SNAP-tag have several disadvantages, especially the long labeling times that might be challenging when co-culture dynamics are studied . Moreover, a bilin-binding fluorescent protein was recently established as a fluorescent reporter, which is functional under anaerobic conditions and might be combined with FAST for multicolor approaches. We showed that the two reporter proteins greenFAST and redFAST are suitable for use in anaerobic synthetic co-culture experiments to detect protein-producing cells as fluorescence is fast, reversible, and causes strong fluorescence.…”

Section: Resultsmentioning

confidence: 99%

Establishment of Green- and Red-Fluorescent Reporter Proteins Based on the Fluorescence-Activating and Absorption-Shifting Tag for Use in Acetogenic and Solventogenic Anaerobes

et al. 2022

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Anaerobic bacteria are promising biocatalysts to produce industrially relevant products from nonfood feedstocks. Several anaerobes are genetically accessible, and various molecular tools for metabolic engineering are available. Still, the use of bright fluorescent reporters, which are commonly used in molecular biological approaches is limited under anaerobic conditions. Therefore, the establishment of different anaerobic fluorescent reporter proteins is of great interest. Here, we present the establishment of the green-and red-fluorescent reporter proteins greenFAST and redFAST for use in different solventogenic and acetogenic bacteria. Green fluorescence of greenFAST was bright in Clostridium saccharoperbutylacetonicum, Clostridium acetobutylicum, Acetobacterium woodii, and Eubacterium limosum, while only C. saccharoperbutylacetonicum showed bright red fluorescence when producing redFAST. We used both reporter proteins in C. saccharoperbutylacetonicum for multicolor approaches. These include the investigation of the co-culture dynamics of metabolically engineered strains. Moreover, we established a tightly regulated inducible two-plasmid system and used greenFAST and redFAST to track the coexistence and interaction of both plasmids under anaerobic conditions in C. saccharoperbutylacetonicum. The establishment of greenFAST and redFAST as fluorescent reporters opens the door for further multicolor approaches to investigate cell dynamics, gene expression, or protein localization under anaerobic conditions.

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“…It is pointed out that not all fluorescent proteins are well folded in E. coli. Soluble production of the bilin-binding fluorescent protein like UnaG-bilirubin as a noncovalent ligand-dependent reporter requires fusion of the solubility enhancer GST or MBP [42,43].…”

Section: Discussionmentioning

confidence: 99%

Detection of human annexin A1 as the novel N-terminal tag for separation and purification handle

Zhang

Dang

et al. 2023

Microb Cell Fact

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Background Several fusion tags for separation handle have been developed, but the fused tag for simply and cheaply separating the target protein is still lacking. Results Separation conditions for the human annexin A1 (hanA1) tagged emerald green fluorescent protein (EmGFP) in Escherichia coli were optimized via precipitation with calcium chloride (CaCl2) and resolubilization with ethylenediamine tetraacetic acid disodium salt (EDTA-Na2). The HanA1-EmGFP absorbing with other three affinity matrix was detected, only it was strongly bound to heparin Sepharose. The separation efficiency of the HanA1-EmGFP was comparable with purification efficiency of the His6-tagged HanA1-EmGFP via metal ion affinity chromatography. Three fluorescent proteins for the EmGFP, mCherry red fluorescent protein and flavin-binding cyan-green fluorescent protein LOV from Chlamydomonas reinhardtii were used for naked-eye detection of the separation and purification processes, and two colored proteins including a red protein for a Vitreoscilla hemoglobin (Vhb), and a brown protein for maize sirohydrochlorin ferrochelatase (mSF) were used for visualizing the separation process. The added EDTA-Na2 disrupted the Fe–S cluster in the mSF, but it showed little impact on heme in Vhb. Conclusions The selected five colored proteins were efficient for detecting the applicability of the highly selective hanA1 for fusion separation and purification handle. The fused hanA1 tag will be potentially used for simple and cheap affinity separation of the target proteins in industry and diagnosis.

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New Orange Ligand-Dependent Fluorescent Reporter for Anaerobic Imaging

Cited by 11 publications

References 34 publications

Double emulsions as a high-throughput enrichment and isolation platform for slower-growing microbes

Double emulsions as a high-throughput enrichment and isolation platform for slower-growing microbes

Establishment of Green- and Red-Fluorescent Reporter Proteins Based on the Fluorescence-Activating and Absorption-Shifting Tag for Use in Acetogenic and Solventogenic Anaerobes

Detection of human annexin A1 as the novel N-terminal tag for separation and purification handle

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