Scaling production of GFP1-10 detector protein in E. coli for secretion screening by split GFP assay | Microbial Cell Factories | Full Text
Background The split GFP assay is a well-known technology for activity-independent screening of target proteins. A superfolder GFP is split into two non-fluorescent parts, GFP11 which is fused to the target protein and GFP1-10. In the presence of both, GFP1-10 and the GFP11-tag are self-assembled and a functional chromophore is formed. However, it relies on the availability and quality of GFP1-10 detector protein to develop fluorescence by assembly with the GFP11-tag connected to the target protein.
GFP1-10 detector protein is often produced in small scale shake flask cultivation and purified from inclusion bodies. Results The production of GFP1-10 in inclusion bodies and purification was comprehensively studied based on Escherichia coli as host. Cultivation in complex and defined medium as well as different feed strategies were tested in laboratory-scale bioreactor cultivation and a standardized process was developed providing high quantity of GFP1-10 detector protein with suitable quality. Split GFP assay was standardized to obtain robust and reliable assay results from cutinase secretion strains of Corynebacterium glutamicum with Bacillus subtilis Sec signal peptides NprE and Pel.
Influencing factors from environmental conditions, such as pH and temperature were thoroughly investigated. Conclusions GFP1-10 detector protein production could be successfully scaled from shake flask to laboratory scale bioreactor. A single run yielded sufficient material for up to 385 96-well plate screening runs. The application study with cutinase secretory strains showed very high correlation between measured cutinase activity to split GFP fluorescence signal proofing applicability for larger screening studies.
A vast microbial community inhabits in the rhizosphere, among which, specialized bacteria known as Plant Growth-Promoting Rhizobacteria (PGPR) confer benefits to host plants including growth promotion and disease suppression. PGPR taxa vary in the ways whereby they curtail the negative effects of invading plant pathogens. However, a cumulative or synergistic effect does not always ensue when a bacterial consortium is used. In this review, we reassess the disease-suppressive mechanisms of PGPR and present explanations and illustrations for functional diversity and/or stability among PGPR taxa regarding these mechanisms.
We also provide evidence of benefits when PGPR mixtures, rather than individuals, are used for protecting crops from various diseases, and underscore the critical determinant factors for successful use of PGPR mixtures. Then, we evaluate the challenges of and limitations to achieving the desired outcomes from strain/species-rich bacterial assemblages, particularly in relation to their role for plant disease management. In addition, towards locating additive or synergistic outcomes, we highlight why and how the benefits conferred need to be categorized and quantified when different strains/species of PGPR are used in combinations. Finally, we highlight the critical approaches needed for developing PGPR mixtures with improved efficacy and stability as biocontrols for utilization in agricultural fields.