The Impact of IPTG Induction on Plasmid Stability and Heterologous Protein Expression by Escherichia coli Biofilms

Gomes, Luciana C.; Monteiro, Gabriel A.; Mergulhão, Filipe

doi:10.3390/ijms21020576

Cited by 44 publications

(33 citation statements)

References 45 publications

(76 reference statements)

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“…This trend is counterintuitive given that an increasing IPTG concentrations will cause expression of the GOI and any L2 regulators, increasing the burden on the cell. However, it is known that IPTG can have unexpected effects on cell physiology 39 and cause changes in plasmid stability 40 , which could lead to reduced overall burden due to fewer copies of the controller plasmid or more efficient utilisation of available nutrients by the cell.…”

Section: Resultsmentioning

confidence: 99%

Harnessing the central dogma for stringent multi-level control of gene expression

et al. 2021

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Strictly controlled inducible gene expression is crucial when engineering biological systems where even tiny amounts of a protein have a large impact on function or host cell viability. In these cases, leaky protein production must be avoided, but without affecting the achievable range of expression. Here, we demonstrate how the central dogma offers a simple solution to this challenge. By simultaneously regulating transcription and translation, we show how basal expression of an inducible system can be reduced, with little impact on the maximum expression rate. Using this approach, we create several stringent expression systems displaying >1000-fold change in their output after induction and show how multi-level regulation can suppress transcriptional noise and create digital-like switches between ‘on’ and ‘off’ states. These tools will aid those working with toxic genes or requiring precise regulation and propagation of cellular signals, plus illustrate the value of more diverse regulatory designs for synthetic biology.

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Section: Resultsmentioning

confidence: 99%

Harnessing the central dogma for stringent multi-level control of gene expression

et al. 2021

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“…Recently the effect of IPTG, which is used to induce production of HemA in our complemented strains, has been shown to negatively affect plasmid stability in, and viability of, planktonically growing E. coli cells [32]. This makes interpretation of our data difficult as the transition from planktonic to biofilm-growing cells is exactly when we need to induce expression of hemA with IPTG to determine its effect on biofilm formation in our complemented strains.…”

Section: Discussionmentioning

confidence: 99%

A novel hemA mutation is responsible for a small-colony-variant phenotype in Escherichia coli

2021

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We identified a small colony variant (SCV) of an amoxicillin/clavulanic acid-resistant derivative of a clinical isolate of Escherichia coli from Malawi, which was selected for in vitro in a subinhibitory concentration of gentamicin. The SCV was auxotrophic for hemin and had impaired biofilm formation compared to the ancestral isolates. A single novel nucleotide polymorphism (SNP) in hemA, which encodes a glutamyl-tRNA reductase that catalyses the initial step of porphyrin biosynthesis leading to the production of haem, was responsible for the SCV phenotype. We showed the SNP in hemA resulted in a significant fitness cost to the isolate, which persisted even in the presence of hemin. However, the phenotype quickly reverted during sequential sub-culturing in liquid growth media. As hemA is not found in mammalian cells, and disruption of the gene results in a significant fitness cost, it represents a potential target for novel drug development specifically for the treatment of catheter-associated urinary tract infections caused by E. coli .

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“…This is to model variation between replicates, and the discrepancies between the estimated and the actual opening energies in vivo . For each round of transcription, mRNA copies were randomly generated from 30 to 60 plasmid DNA copies ( 3 , 68 , 69 ). We chose an optimum opening energy of 12 kcal/mol or less for translation.…”

Section: Methodsmentioning

confidence: 99%

Protein yield is tunable by synonymous codon changes of translation initiation sites

Bhandari

Lim

Remus

et al. 2019

Preprint

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149/150 words) Recombinant protein production in microbial systems is well-established, yet half of these experiments have failed in the expression phase. Failures are expected for 'difficult-to-express' proteins, but for others, codon bias, mRNA folding, avoidance, and G+C content have been suggested to explain observed levels of protein expression. However, determining which of these is the strongest predictor is still an active area of research. We used an ensemble average of energy model for RNA to show that the accessibility of translation initiation sites outperforms other features in predicting the outcomes of 11,430 experiments of recombinant protein production in Escherichia coli . We developed TIsigner and showed that synonymous codon changes within the first nine codons are sufficient to improve the accessibility of translation initiation sites. Our software produces scores for both input and optimised sequences, so that success/failure can be predicted and prevented by PCR cloning of optimised sequences.

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The Impact of IPTG Induction on Plasmid Stability and Heterologous Protein Expression by Escherichia coli Biofilms

Cited by 44 publications

References 45 publications

Harnessing the central dogma for stringent multi-level control of gene expression

Harnessing the central dogma for stringent multi-level control of gene expression

A novel hemA mutation is responsible for a small-colony-variant phenotype in Escherichia coli

Protein yield is tunable by synonymous codon changes of translation initiation sites

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