pTRA – A reporter system for monitoring the intracellular dynamics of gene expression

Wagner, Sabine G.; Ziegler, Martin; Löwe, Hannes; Kremling, Andreas; Pflüger‐Grau, Katharina

doi:10.1371/journal.pone.0197420

Cited by 1 publication

(4 citation statements)

References 14 publications

(16 reference statements)

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“…This relation was analysed by growing E . coli HMS174/DE3 wildtype and the same strain carrying the plasmid pTRA-51hd [46]. Via dosing defined substrate uptake rates the related growth rates were determined.…”

Section: Resultsmentioning

confidence: 99%

“…coli species used in this work was the strain HMS174/DE3 [47]. To provoke a burden (shown in Fig 8), the inducible plasmid pTRA-51hd was inserted [46]. All pre-cultures of E .…”

Section: Methodsmentioning

confidence: 99%

“…All pre-cultures of E . coli cells were cultivated first in lysogeny broth (LB) medium [48] and then in minimal medium [46] amended with trace elements, and 20 mM of glucose as C- source. The pH was set to 7.4 (25°C) and a salt solution as well as trace elements were added.…”

Section: Methodsmentioning

confidence: 99%

“…The pH was set to 7.4 (25°C) and a salt solution as well as trace elements were added. Pre-cultures were grown the same way as described in [46], before they were used to inoculate 50 mL of the minimal medium mentioned above without C-source to a cell-dry-weight-concentration of around 0.04 mg mL -1 . The principle cultivation setup is depicted in Fig 7A.…”

Section: Methodsmentioning

confidence: 99%

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An automated and parallelised DIY-dosing unit for individual and complex feeding profiles: Construction, validation and applications

et al. 2019

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Since biotechnological research becomes more and more important for industrial applications, there is an increasing need for scalable and controllable laboratory procedures. A widely used approach in biotechnological research to improve the performance of a process is to vary the growth rates in order to find the right balance between growth and the production. This can be achieved by the application of a suitable feeding strategy. During this initial bioprocess development, it is beneficial to have at hand cheap and easy setups that work in parallel (e.g. in shaking flasks). Unfortunately, there is a gap between these easy setups and defined and controllable processes, which are necessary for up-scaling to an industrial relevant volume. One prerequisite to test and evaluate different process strategies apart from batch-mode is the availability of pump systems that allow for defined feeding profiles in shaking flasks. To our knowledge, there is no suitable dosing device on the market which fulfils the requirements of being cheap, precise, programmable, and parallelizable. Commercially available dosing units are either already integrated in bioreactors and therefore inflexible, or not programmable, or expensive, or a combination of those. Here, we present a LEGO-MINDSTORMS-based syringe pump, which has the potential of being widely used in daily laboratory routine due to its low price, programmability, and parallelisability. The acquisition costs do not exceed 350 € for up to four dosing units, that are independently controllable with one EV3 block. The system covers flow rates ranging from 0.7 μL min -1 up to 210 mL min -1 with a reliable flux. One dosing unit can convey at maximum a volume of 20 mL (using all 4 units even up to 80 mL in total) over the whole process time. The design of the dosing unit enables the user to perform experiments with up to four different growth rates in parallel (each measured in triplicates) per EV3-block used. We estimate, that the LEGO-MINDSTORMS-based dosing unit with 12 syringes in parallel is reducing the costs up to 50-fold compared to a trivial version of a commercial pump system (~1500 €) which fits the same requirements. Using the pump, we set the growth rates of a E . coli HMS174/DE3 culture to values between 0.1 and 0.4 h -1 with a standard deviation of at best 0.35% and an average discrepancy of 13.2%. Additionally, we determined the energy demand of a culture for the maintenance of the pTRA-51hd plasmid by quantifying the changes in biomass yield with different growth rates set. Around 25% of total substrate taken up is used for plasmid maintenance. To present possible applications and show the flexibility of the system, we applied a constant feed to perform microencapsulation of Pseudomonas putida and an individual dosing profile for the purification of a his-tagged eGFP via IMAC. This smart and versatile dosing unit, which is ...

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Section: Resultsmentioning

confidence: 99%

“…coli species used in this work was the strain HMS174/DE3 [47]. To provoke a burden (shown in Fig 8), the inducible plasmid pTRA-51hd was inserted [46]. All pre-cultures of E .…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations