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000865570 1001_ $$0P:(DE-Juel1)165764$$aOtten, Julia$$b0
000865570 245__ $$aA FRET-based biosensor for the quantification of glucose in culture supernatants of mL scale microbial cultivations
000865570 260__ $$aLondon$$bBiomed Central$$c2019
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000865570 520__ $$aBackgroundIn most microbial cultivations d-glucose is the main carbon and energy source. However, quantification of d-glucose especially in small scale is still challenging. Therefore, we developed a FRET-based glucose biosensor, which can be applied in microbioreactor-based cultivations. This sensor consists of a glucose binding protein sandwiched between two fluorescent proteins, constituting a FRET pair. Upon d-glucose binding the sensor undergoes a conformational change which is translated into a FRET-ratio change.ResultsThe selected sensor shows an apparent Kd below 1.5 mM d-glucose and a very high sensitivity of up to 70% FRET-ratio change between the unbound and the glucose-saturated state. The soluble sensor was successfully applied online to monitor the glucose concentration in an Escherichia coli culture. Additionally, this sensor was utilized in an at-line process for a Corynebacterium glutamicum culture as an example for a process with cell-specific background (e.g. autofluorescence) and medium-induced quenching. Immobilization of the sensor via HaloTag® enabled purification and covalent immobilization in one step and increased the stability during application, significantly.ConclusionA FRET-based glucose sensor was used to quantify d-glucose consumption in microtiter plate based cultivations. To the best of our knowledge, this is the first method reported for online quantification of d-glucose in microtiter plate based cultivations. In comparison to d-glucose analysis via an enzymatic assay and HPLC, the sensor performed equally well, but enabled much faster measurements, which allowed to speed up microbial strain development significantly.
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000865570 7001_ $$0P:(DE-Juel1)168172$$aTenhaef, Niklas$$b1
000865570 7001_ $$0P:(DE-Juel1)171232$$aJansen, Roman P.$$b2
000865570 7001_ $$0P:(DE-Juel1)166538$$aDöbber, Johannes$$b3
000865570 7001_ $$0P:(DE-Juel1)177977$$aJungbluth, Lisa$$b4
000865570 7001_ $$0P:(DE-Juel1)129050$$aNoack, Stephan$$b5
000865570 7001_ $$0P:(DE-Juel1)129053$$aOldiges, Marco$$b6
000865570 7001_ $$0P:(DE-Juel1)129076$$aWiechert, Wolfgang$$b7
000865570 7001_ $$0P:(DE-Juel1)131522$$aPohl, Martina$$b8$$eCorresponding author
000865570 773__ $$0PERI:(DE-600)2091377-1$$a10.1186/s12934-019-1193-y$$gVol. 18, no. 1, p. 143$$n1$$p143$$tMicrobial cell factories$$v18$$x1475-2859$$y2019
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