High-level homogeneous production of fluorescent proteins in E. coli provides a sensitive reporter for antibiotic activity detection

Baranova, M.; Terekhov, S.; Mokrushina, Y.; Smirnov, I.V.

Авторы: Baranova M., Terekhov S., Mokrushina Y., Smirnov I.V.
Журнал: FEBS open bio
Номер: 11
Год издания: 2021
Издательство: John Wiley & Sons Inc.
Местоположение издательства: United States
Первая страница: 151
DOI: 10.1002/2211-5463.13205
Аннотация: The uncontrolled use of antibiotics both in the medicinal practiceand agriculture demands the development of highly sensitiveprobes for their preventive detection. Otherwise, this misuseresults in the global spread of multidrug-resistant strains. That isparticularly critical in the case of gram-negative bacteria whichare intrinsically resistant to the majority of novel antimicrobialagents. Recently we presented a concept of deep functional pro-ﬁling of microbiomes providing a new powerful tool for thedetection of biological activity of even minor components of bac-terial communities. This concept is based on a combination ofmicroﬂuidic encapsulation of single cells in droplets of doubleemulsion (MDE) and ﬂuorescence-activated cell sorting (FACS)isolating phenotype of interest. The stumbling block of this tech-nology is the requirement of a particular ﬂuorescent reporterindicating the desired activity. Moreover, highly sensitive detec-tion of antibiotics in various environments is an urgent taskresulted from their uncontrolled use in medicinal practice andagriculture. The present study is dedicated to the development ofan Escherichia coli strain as a Gram-negative reporter. We havetested a number of common laboratory E. coli strains for optimalgrowth conditions, cell morphology, and culture homogeneity.Different bright GFP derivative genes regulated by variousstrong promoters were examined to gain high levels of produc-tion, signal intensity, and narrow ﬂuorescence distribution. Bulkculture ﬂuorescence measurements supplemented with ﬂuorescentmicroscopy and ﬂow cytometry allowed us to identify a combina-tion of strain, gene, and promoter providing optimal biosensorparameters. We believe our results will be applied for variousapplications including food safety-related studies, antibiotic pol-lutant tracking, and antibiotic discovery. This work was sup-ported by RSF grant 19-14-00331
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