title: Making Fluorescent Streptococci and Enterococci for Live Imaging.
abstract: Since the discovery of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria, outstanding fluorescent labeling tools with numerous applications in vastly different areas of life sciences have been developed. To optimize GFP for diverse life science applications, a large variety of GFP derivatives with different environmental characteristics have been generated by mutagenesis. The enhanced green fluorescent protein (EGFP) is a well-known GFP derivative with highly increased fluorescence intensity compared to the GFP wild-type molecule. Further optimization strategies include numerous GFP derivatives with blue- and yellow-shifted fluorescence and increased pH-stability. The methods reported herein describe in detail the construction of customized fluorescent GFP reporter plasmids where the fluorescence gene is expressed under the control of a certain bacterial promoter of interest. Special attention is given to the GFP derivatives EGFP and Sirius. We explain how to generate EGFP/Sirius expressing streptococci and how to employ recombinantly labeled streptococci in different downstream fluorescent applications.
authors.affiliation: Institute of Medical Microbiology and Hygiene, University of Ulm, Albert-Einstein-Allee 11, 89081, Ulm, Germany.|Faculty of Pharmacy, Department of Microbiology and Immunology, Suez Canal University, Ismailia, Egypt.
authors.forename: Sarah
authors.initials: S
authors.lastname: Shabayek
authors.affiliation: Institute of Medical Microbiology and Hygiene, University of Ulm, Albert-Einstein-Allee 11, 89081, Ulm, Germany. barbara.spellerberg@uniklinik-ulm.de.
authors.forename: Barbara
authors.initials: B
authors.lastname: Spellerberg
mesh_terms: D002460:Cell Line; D003001:Cloning, Molecular; D016983:Enterococcus / Q000235:genetics* / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D008264:Macrophages / Q000382:microbiology; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging / Q000379:methods; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D013291:Streptococcus / Q000235:genetics* / Q000378:metabolism; D014169:Transformation, Bacterial
chemical_list: D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins
keywords: EGFP; Fluorescence labeling; GFP; Green fluorescent protein; Sirius; Streptococci