No abstract

Background: In industry and academic research, there is an increasing demand for flexible automated microfermentation platforms with advanced sensing technology. However, up to now, conventional platforms cannot generate continuous data in high-throughput cultivations, in particular for monitoring biomass and fluorescent proteins. Furthermore, microfermentation platforms are needed that can easily combine cost-effective, disposable microbioreactors with downstream processing and analytical assays.

In the last decade, quantitative analysis of cellular processes has enabled detailed insights into the complexity of metabolic and regulatory networks in various microorganisms, especially in the fields of systems and synthetic biology. Within the respective research areas, fluorescent proteins (FPs) are used as versatile in vivo reporters to study gene regulation and protein synthesis, folding, localization, and activity (1, 5, 7) in bacteria and yeast. The most widely used FPs are the green fluorescent protein (GFP) and its derivatives. However, their use as in vivo reporter proteins is restricted by various environmental factors affecting their signal intensity, including the availability of oxygen during maturation (18,20). In addition, time-consuming protein folding and chromophore formation cause a time lag between the detectable fluorescence signal and the amount of reporter protein produced at a certain time point. Recently, we have developed an alternative family of fluorescent proteins that binds flavin mononucleotide (FMN) as a chromophore (3, 4). In contrast to all members of the GFP family, these novel FMN-binding fluorescent proteins (FbFPs) exhibit bright cyan-green fluorescence under both aerobic and anaerobic conditions and can be used to label facultative and strict anaerobic bacteria (3, 16) and yeast (19).Here, we comparatively analyzed whether FbFP and a GFP-derived enhanced yellow fluorescent protein (YFP) can be used as reporters for quantitative real-time analysis of gene expression in living Escherichia coli cells. Furthermore, we studied whether the detectable FP fluorescence intensities correlate with the amounts of reporter proteins continuously during cell growth. Therefore, both FPs were expressed in E. coli using vector pRhokHi-2 (12), which harbors the aphII promoter of the kanamycin resistance gene and allows the constitutive expression of both fluorescence proteins at moderate levels. The pRhokHi-2-YFP expression vector, encoding yellow fluorescent GFP-10C derivative YFP (15) (available from Clontech-Takara Bio Europe, Saint-Germain-en-Laye, France), was constructed as described by Katzke et al. (12). Expression vector pRhokHi-2-FbFP, encoding E. coli FbFP (EcFbFP) (GenBank accession number ABN71355) (evoglow-Bs2; evocatal GmbH, Düsseldorf, Germany), was generated by PCR amplification of the fluorescence reporter gene, while NdeI and XhoI restriction sites were introduced into its 5Ј and 3Ј ends, respectively. Subsequently, the reporter gene was cloned into the corresponding sites of the expression vector pRhokHi-2. P aphII -dependent FP expression basically avoids protein misfolding caused by high-level gene expression and alterations of FP expression levels due to changes in the inducer-to-cell ratio during cultivation.In order to continuously monitor FP expression and in vivo fluorescence, E. coli DH5␣ (8) strains carrying the FP-encoding expression vector pRhokHi-2-YFP or pRhokHi-2-FbFP were generated. The respective empty vector was used as a negative control. Subsequently,...

No abstract

Please note that this is an author-produced PDF of an article accepted for publication following peer review. The definitive publisher-authenticated version is available on the publisher Web site Highlights ► Societies increasingly depend on timely information on ecosystems and natural hazards. ► Data is needed to improve climate-related uncertainty and geo-hazard early warning. ► Observatory networks coordinate and integrate the collection of standardised data. ► Ocean observatories provide opportunity for ocean science to evolve.