scholarly journals Chromatic bacteria – A broad host-range plasmid and chromosomal insertion toolbox for fluorescent protein expression in bacteria

2018 ◽  
Author(s):  
Rudolf O. Schlechter ◽  
Hyunwoo Jun ◽  
Michał Bernach ◽  
Simisola Oso ◽  
Erica Boyd ◽  
...  
Keyword(s):  
Host Range ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Broad Host Range ◽  
Complex Environments ◽  
Chloramphenicol Resistance ◽  
Red Fluorescent Proteins ◽  
Broad Host Range Plasmid ◽  
Environmental Interactions ◽  
Yellow Orange

AbstractDifferential fluorescent labelling of bacteria has become instrumental for many aspects of microbiological research, such as the study of biofilm formation, bacterial individuality, evolution, and bacterial behaviour in complex environments. We designed a variety of plasmids, each bearing one of eight unique, constitutively expressed fluorescent protein genes in conjunction with one of four different antibiotic resistance combinations. The fluorophores mTagBFP2, mTurquoise2, sGFP2, mClover3, sYFP2, mOrange2, mScarlet-I, and mCardinal, encoding for blue, cyan, green, green-yellow, yellow, orange, red, and far-red fluorescent proteins, respectively, were combined with selectable markers conferring tetracycline, gentamicin, kanamycin, and/or chloramphenicol resistance. These constructs were cloned into three different plasmid backbones: a broad host-range plasmid, a Tn5transposon delivery plasmid, and a Tn7transposon delivery plasmid. The utility of the plasmids and transposons was tested in bacteria from the phyla Actinobacteria, Proteobacteria, and Bacteroidetes. We were able to tag representatives from the phylum Proteobacteria at least via our Tn5transposon delivery system. The here constructed plasmids are available to the community and provide a valuable tool to investigate bacteria-bacteria, bacteria-host, and bacteria-environmental interactions.

scholarly journals Split-wrmScarlet and split-sfGFP: Tools for faster, easier fluorescent l abeling of endogenous proteins in Caenorhabditis elegans

Genetics ◽  
2021 ◽  
Author(s):  
Jérôme Goudeau ◽  
Catherine S Sharp ◽  
Jonathan Paw ◽  
Laura Savy ◽  
Manuel D Leonetti ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Labeling ◽  
Large Fragment ◽  
C Elegans ◽  
High Affinity ◽  
Red Fluorescent Proteins ◽  
Invaluable Tool ◽  
Endogenous Proteins ◽  
Fluorescent Tags

Abstract We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous C. elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663

Broad Host-Range Plasmid R1162: Replication, Incompatibility, and Copy-Number Control

1985 ◽  
pp. 173-188 ◽  
Author(s):  
Richard J. Meyer ◽  
Lung-Shen Lin ◽  
Kyunghoon Kim ◽  
Michael A. Brasch
Keyword(s):  
Host Range ◽  
Copy Number ◽  
Broad Host Range ◽  
Copy Number Control ◽  
Broad Host Range Plasmid

scholarly journals Promoters of the broad host range plasmid RK2: analysis of transcription (initiation) in five species of gram-negative bacteria.

Genetics ◽  
1992 ◽  
Vol 130 (1) ◽  
pp. 27-36 ◽  
Author(s):  
A Greener ◽  
S M Lehman ◽  
D R Helinski
Keyword(s):  
Host Range ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Firefly Luciferase ◽  
Broad Host Range ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Broad Host Range Plasmid ◽  
Transcriptional Start Sites ◽  
Plasmid Rk2

Abstract A broad host range cloning vector was constructed, suitable for monitoring promoter activity in diverse Gram-negative bacteria. This vector, derived from plasmid RSF1010, utilized the firefly luciferase gene as the reporter, since the assay for its bioluminescent product is sensitive, and measurements can be made without background from the host. Twelve DNA fragments with promoter activity were obtained from broad host range plasmid RK2 and inserted into the RSF1010 derived vector. The relative luciferase activities were determined for these fragments in five species of Gram-negative bacteria. In addition, four promoters were analyzed by primer extension to locate transcriptional start sites in each host. The results show that several of the promoters vary substantially in relative strengths or utilize different transcriptional start sites in different bacteria. Other promoters exhibited similar activities and identical start sites in the five hosts examined.

scholarly journals Acclimatization of symbiotic corals to mesophotic light environments through wavelength transformation by fluorescent protein pigments

2017 ◽  
Vol 284 (1858) ◽  
pp. 20170320 ◽  
Author(s):  
Edward G. Smith ◽  
Cecilia D'Angelo ◽  
Yoni Sharon ◽  
Dan Tchernov ◽  
Joerg Wiedenmann
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Environmental Gradients ◽  
Red Light ◽  
Spectral Composition ◽  
Light Environment ◽  
Homogeneous Distribution ◽  
Red Fluorescent Proteins ◽  
Ecological Importance ◽  
Colour Morphs

The depth distribution of reef-building corals exposes their photosynthetic symbionts of the genus Symbiodinium to extreme gradients in the intensity and spectral quality of the ambient light environment. Characterizing the mechanisms used by the coral holobiont to respond to the low intensity and reduced spectral composition of the light environment in deeper reefs (greater than 20 m) is fundamental to our understanding of the functioning and structure of reefs across depth gradients. Here, we demonstrate that host pigments, specifically photoconvertible red fluorescent proteins (pcRFPs), can promote coral adaptation/acclimatization to deeper-water light environments by transforming the prevalent blue light into orange-red light, which can penetrate deeper within zooxanthellae-containing tissues; this facilitates a more homogeneous distribution of photons across symbiont communities. The ecological importance of pcRFPs in deeper reefs is supported by the increasing proportion of red fluorescent corals with depth (measured down to 45 m) and increased survival of colour morphs with strong expression of pcRFPs in long-term light manipulation experiments. In addition to screening by host pigments from high light intensities in shallow water, the spectral transformation observed in deeper-water corals highlights the importance of GFP-like protein expression as an ecological mechanism to support the functioning of the coral– Symbiodinium association across steep environmental gradients.

Sign in / Sign up

Export Citation Format

Share Document