Robustness testing and scalability of phosphate regulated promoters useful for two-stage autoinduction in<i>E. coli</i>

Moreb, Eirik A.; Ye, Zhixia; Efromson, John P.; Hennigan, Jennifer N.; Menacho‐Melgar, Romel; Lynch, Michael

doi:10.1101/2020.01.26.920280

Cited by 4 publications

(2 citation statements)

References 28 publications

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“…To build upon this success and to further our systems engineering approach, we also integrated dynamic control of autolysis enzymes into JNH_Redox6E, culminating in the development of autoDC REdox for subsequent robustness studies 58 . It is worth noting that we use the "yibDp gene" promoter because of its well-characterized robustness 59 .…”

Section: Strain Engineeringmentioning

confidence: 99%

Scalable, Robust, High-throughput Expression, Purification & Characterization of Nanobodies Enabled by 2-Stage Dynamic Control

Hennigan,

Menacho-Melgar,

Sarkar

et al. 2023

Preprint

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Nanobodies are single-domain antibody fragments that have garnered considerable use as diagnostic and therapeutic agents as well as research tools. However, obtaining pure VHHs, like many proteins, can be laborious and inconsistent. High level cytoplasmic expression inE. colican be challenging due to improper folding and insoluble aggregation caused by reduction of the conserved disulfide bond. We report a systems engineering approach leveraging engineered strains ofE. coli, in combination with a two-stage process and simplified downstream purification, enabling improved, robust soluble cytoplasmic nanobody expression, as well as rapid cell autolysis and purification. This approach relies on the dynamic control over the reduction potential of the cytoplasm, in combination with dynamic expression of chaperones and lysis enzymes needed for purification. Collectively, the engineered system results in more robust growth and protein expression, enabling efficient scalable nanobody production, and purification from high throughput microtiter plates, to routine shake flask cultures and larger instrumented bioreactors. We expect this system will expedite VHH development.

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Section: Strain Engineeringmentioning

confidence: 99%

Scalable, Robust, High-throughput Expression, Purification & Characterization of Nanobodies Enabled by 2-Stage Dynamic Control

Hennigan,

Menacho-Melgar,

Sarkar

et al. 2023

Preprint

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“…12 Biosensors used in two-stage dynamic metabolic control have to be optimized to perform under both growth conditions. 39,40 Many MRTF-based biosensors use a repressed-repressor architecture. 41,42 In this architecture, the MRTF represses gene expression from its cognate promoter in the absence of its target metabolite, but its expression repression activity is antagonized by the presence of a specific intracellular metabolite (Figure 1A).…”

mentioning

confidence: 99%

The Growth Dependent Design Constraints of Transcription-Factor-Based Metabolite Biosensors

Hartline

Zhang

2022

ACS Synth. Biol.

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Metabolite biosensors based on metabolite-responsive transcription factors are key synthetic biology components for sensing and precisely controlling cellular metabolism. Biosensors are often designed under laboratory conditions but are deployed in applications where cellular growth rate differs drastically from its initial characterization. Here we asked how growth rate impacts the minimum and maximum biosensor outputs and the dynamic range, which are key metrics of biosensor performance. Using LacI, TetR, and FadR-based biosensors in Escherichia coli as models, we find that the dynamic range of different biosensors have different growth rate dependencies. We developed a kinetic model to explore how tuning biosensor parameters impact the dynamic range growth rate dependence. Our modeling and experimental results revealed that the effects to dynamic range and its growth rate dependence are often coupled, and the metabolite transport mechanisms shape the dynamic range-growth rate response. This work provides a systematic understanding of biosensor performance under different growth rates, which will be useful for predicting biosensor behavior in broad synthetic biology and metabolic engineering applications.

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Scalable, robust, high-throughput expression & purification of nanobodies enabled by 2-stage dynamic control

Hennigan,

Menacho-Melgar,

Sarkar

et al. 2024

Metabolic Engineering

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Robustness testing and scalability of phosphate regulated promoters useful for two-stage autoinduction inE. coli

Cited by 4 publications

References 28 publications

Scalable, Robust, High-throughput Expression, Purification & Characterization of Nanobodies Enabled by 2-Stage Dynamic Control

Scalable, Robust, High-throughput Expression, Purification & Characterization of Nanobodies Enabled by 2-Stage Dynamic Control

The Growth Dependent Design Constraints of Transcription-Factor-Based Metabolite Biosensors

Scalable, robust, high-throughput expression & purification of nanobodies enabled by 2-stage dynamic control

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