Overloaded and stressed: whole-cell considerations for bacterial synthetic biology

Borkowski, Olivier; Ceroni, Francesca; Stan, Guy-Bart; Ellis, Tom

doi:10.1016/j.mib.2016.07.009

Cited by 219 publications

(218 citation statements)

References 41 publications

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“…As we try to build synthetic gene regulation networks, we need to understand competition for resources giving rise to limitations and tradeoffs. Both mathematical modeling and experimental studies are directed towards elucidation of this issue [17,18,7]. In the system studied, we provide evidence that suppression of gene expression due to added NTP is not primarily due to competition between transcription and translation as has been proposed in [7], but we suggest that the limitation of available free Mg 2+ plays a role in this suppression.…”

Section: +supporting

confidence: 53%

Translation Inhibition and Resource Balance in the Cell-Free Gene Expression System

Nagaraj

Greene

Sengupta

et al. 2017

Preprint

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Quantifying the effect of vital resources on transcription and translation helps to understand the degree to which the concentration of each resource must be regulated for achieving homeostasis. Utilizing the synthetic transcription-translation (TX-TL) system, we study the impact of nucleotide triphosphates (NTPs) and magnesium (Mg 2+ ), on gene expression. Recent observations of the counterintuitive phenomenon of suppression of gene expression at high NTP concentrations have led to the speculation that such suppression is due to the consumption of resources by transcription, hence leaving fewer resources for translation. In this work, we investigate an alternative hypothesis: direct suppression of the translation rate via stoichiometric mismatch in necessary reagents. We observe NTP-dependent suppression even in the early phase of gene expression, contradicting the resource limitation argument. To further decouple the contributions of transcription and translation, we performed gene expression experiments with purified mRNA. Simultaneously monitoring mRNA and protein abundances allowed us to extract a time-dependent translation rate. Measuring translation rates for different Mg 2+ and NTP concentrations, we observe a complex resource dependence. We demonstrate that translation is the rate-limiting process that is directly inhibited by high NTP concentrations. Additional Mg 2+ can partially reverse this inhibition. In several experiments, we observe two maxima of the translation rate viewed as a function of both Mg 2+ and NTP concentration, which can be explained in terms of an NTP-independent effect on the ribosome complex and an NTP-Mg2 + titration effect. The non-trivial compensatory effects of abundance of different vital resources signals the presence of complex regulatory mechanisms to achieve optimal gene expression.

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Section: +supporting

confidence: 53%

Translation Inhibition and Resource Balance in the Cell-Free Gene Expression System

Nagaraj

Greene

Sengupta

et al. 2017

Preprint

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“…Furthermore, its availability as SEVA plasmids eases the reuse of the same plasmids in a variety of Gram‐negative bacteria. Finally, the poor performance of the high PCN construct at 42 °C is a warning about the pursuit of the highest expression levels against the background of the host's retroactivity . In any case, the work above accredits the value of combining improved hosts with matching genetic devices for the sake of building more robust Cell Factories.…”

Section: Discussionmentioning

confidence: 99%

Improved Thermotolerance of Genome‐Reduced Pseudomonas putida EM42 Enables Effective Functioning of the P_L/cI857 System

Aparicio

Lorenzo

Martínez‐García

2018

Biotechnology Journal

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The higher intracellular ATP levels of genome‐edited strains of P. putida that result from deleting various energy‐consuming functions has been exploited for expanding the window of thermal tolerance of this bacterium. Unlike instant growth halt and eventual death of the naturally occurring strain P. putida KT2440 at 42 °C, the EM42 variant maintained growth and viability of most of the population at the higher temperature for at least 6 h. The authors took advantage of this quality for implementing a robust thermo‐inducible heterologous expression device in this species. To this end, the cI857/PL pair of the lambda phage of Escherichia coli was reshaped as a functional cargo that followed the SEVA (Standard European Vector Architecture) format. Quantitation of the transcriptional output of the resulting expression device with GFP reporter technology in various gene dosages identified conditions of unprecedented induced/uninduced ratios (>300 folds) and very high total transcriptional capacity in this bacterial host. The broad‐host range nature of the cognate replication origins makes expression vectors pSEVA2214 (low plasmid copy number), pSEVA2314 (medium), and pSEVA2514 (high) to cover a wide range of heterologous expression needs in P. putida and possibly other Gram‐negative species.

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“…Some degree of burden is expected to accompany heterologous protein expression 28,29 . To assess the burden on EcN cells presented by ARG1, we characterized their growth, viability, reporter expression maintenance, and release of microcins.…”

mentioning

confidence: 99%

Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts

Bourdeau

Lee‐Gosselin

Lakshmanan

et al. 2018

Nature

277

323

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Overloaded and stressed: whole-cell considerations for bacterial synthetic biology

Cited by 219 publications

References 41 publications

Translation Inhibition and Resource Balance in the Cell-Free Gene Expression System

Translation Inhibition and Resource Balance in the Cell-Free Gene Expression System

Improved Thermotolerance of Genome‐Reduced Pseudomonas putida EM42 Enables Effective Functioning of the P_L/cI857 System

Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts

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Overloaded and stressed: whole-cell considerations for bacterial synthetic biology

Cited by 219 publications

References 41 publications

Translation Inhibition and Resource Balance in the Cell-Free Gene Expression System

Translation Inhibition and Resource Balance in the Cell-Free Gene Expression System

Improved Thermotolerance of Genome‐Reduced Pseudomonas putida EM42 Enables Effective Functioning of the PL/cI857 System

Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts

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Product

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Improved Thermotolerance of Genome‐Reduced Pseudomonas putida EM42 Enables Effective Functioning of the P_L/cI857 System