RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution

Kopsidas, George; Carman, Rachael K; Stutt, Emma L; Raicevic, Anna; Roberts, Anthony; Siomos, Mary-Anne; Dobric, Nada; Pontes-Braz, Luisa; Coia, Gregory

doi:10.1186/1472-6750-7-18

Cited by 19 publications

(10 citation statements)

References 42 publications

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“…Substitution S268G is far less common among clinical isolates (present only in TEM‐49 and TEM‐136). This substitution was found in five different experimental studies: four using cefotaxime as a selective agent (Holloway et al , 2007; Kopsidas et al , 2007; Bershtein & Tawfik, 2008; M.L.M. Salverda & J.A.G.M.…”

Section: Comparison Of the Natural And Laboratory Evolution Of Tem‐1mentioning

confidence: 86%

“…The detailed description of the natural evolution of TEM alleles, combined with the experimental ease of selecting for increased antibiotic resistance, has made TEM-1 a frequently used tool for exploring the potential of novel protein engineering techniques (Stemmer, 1994;Zaccolo & Gherardi, 1999;Long-McGie et al, 2000;Camps et al, 2003;Fujii et al, 2004;Kopsidas et al, 2007). Soon, the potential of using in vitro evolution to repeat or even predict the natural evolution of TEM-1 was recognized (Vakulenko et al, 1998), and, by adjusting the mutational spectrum and enzyme expression levels, in vitro evolution protocols were further fine tuned in order to mimic the natural evolutionary process (Barlow & Hall, 2002).…”

Section: Introductionmentioning

confidence: 99%

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Natural evolution of TEM-1 β-lactamase: experimental reconstruction and clinical relevance

2010

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TEM-1 β-lactamase is one of the most well-known antibiotic resistance determinants around. It confers resistance to penicillins and early cephalosporins and has shown an astonishing functional plasticity in response to the introduction of novel drugs derived from these antibiotics. Since its discovery in the 1960s, over 170 variants of TEM-1 - with different amino acid sequences and often resistance phenotypes - have been isolated in hospitals and clinics worldwide. Next to this well-documented 'natural' evolution, the in vitro evolution of TEM-1 has been the focus of attention of many experimental studies. In this review, we compare the natural and laboratory evolution of TEM-1 in order to address the question to what extent the evolution of antibiotic resistance can be repeated, and hence might have been predicted, under laboratory conditions. We also use the comparison to gain an insight into the adaptive relevance of hitherto uncharacterized substitutions present in clinical isolates and to predict substitutions not yet observed in nature. Based on new structural insights, we review what is known about substitutions in TEM-1 that contribute to the extension of its resistance phenotype. Finally, we address the clinical relevance of TEM alleles during the past decade, which has been dominated by the emergence of another β-lactamase, CTX-M.

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Section: Comparison Of the Natural And Laboratory Evolution Of Tem‐1mentioning

confidence: 86%

Section: Introductionmentioning

confidence: 99%

Natural evolution of TEM-1 β-lactamase: experimental reconstruction and clinical relevance

2010

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“…The low fidelity of the MS2 replicase makes it suitable for direct in vivo or in vitro generation of RNA libraries for protein or aptamer selection from clonal RNA templates without the need for DNA mutagenesis. A study describing the in vivo use of Qβ replicase to generate mRNA libraries showed that the mutational spectrum of phage RNA replicases is close to the ideal (67). Furthermore, the independence of the MS2 RdTT system from DNA might increase the repertoire of selection strategies for DNA-modifying enzymes such as DNA nucleases, ligases, polymerases, recombinases or methyltransferases whose activity can interfere with a conventional DNA construct.…”

Section: Discussionmentioning

confidence: 99%

Cell-free expression of RNA encoded genes using MS2 replicase

Weise

Heymann

Mayr

et al. 2019

Nucleic Acids Research

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RNA replicases catalyse transcription and replication of viral RNA genomes. Of particular interest for in vitro studies are phage replicases due to their small number of host factors required for activity and their ability to initiate replication in the absence of any primers. However, the requirements for template recognition by most phage replicases are still only poorly understood. Here, we show that the active replicase of the archetypical RNA phage MS2 can be produced in a recombinant cell-free expression system. We find that the 3′ terminal fusion of antisense RNAs with a domain derived from the reverse complement of the wild type MS2 genome generates efficient templates for transcription by the MS2 replicase. The new system enables DNA-independent gene expression both in batch reactions and in microcompartments. Finally, we demonstrate that MS2-based RNA-dependent transcription-translation reactions can be used to control DNA-dependent gene expression by encoding a viral DNA-dependent RNA polymerase on a MS2 RNA template. Our study sheds light on the template requirements of the MS2 replicase and paves the way for new in vitro applications including the design of genetic circuits combining both DNA- and RNA-encoded systems.

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“…M182T is a stabilizing mutation [28] that is found clinically in resistant alleles to a wide variety of -lactamase substrates and inhibitors but never appears alone [43]. In most directed evolution experiments for increased resistance to cefotaxime, M182T appears after G238S [44][45][46][47][48][49]. M182T-induced stabilization has been shown to have compensatory effects for a wide variety of mutations for amoxicillin resistance [22].…”

Section: S3mentioning

confidence: 99%

Shifting Fitness and Epistatic Landscapes Reflect Trade-offs along an Evolutionary Pathway

Steinberg

Ostermeier

2016

Journal of Molecular Biology

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Nature repurposes proteins via evolutionary processes. Such adaptation can come at the expense of the original protein's function, which is a trade-off of adaptation. We sought to examine other potential adaptive trade-offs. We measured the effect on ampicillin resistance of ~12,500 unique single amino acid mutants of the TEM-1, TEM-17, TEM-19, and TEM-15 β-lactamase alleles, which constitute an adaptive path in the evolution of cefotaxime resistance. These protein fitness landscapes were compared and used to calculate epistatic interactions between these mutations and the two mutations in the pathway (E104K and G238S). This series of protein fitness landscapes provides a systematic, quantitative description of pairwise/tertiary intragenic epistasis involving adaptive mutations. We find that the frequency of mutations exhibiting epistasis increases along the evolutionary pathway. Adaptation moves the protein to a region in the fitness landscape characterized by decreased mutational robustness and increased ruggedness, as measured by fitness effects of mutations and epistatic interactions for TEM-1's original function. This movement to such a "fitness territory" has evolutionary consequences and is an important adaptive trade-off and cost of adaptation. Our systematic study provides detailed insight into the relationships between mutation, protein structure, protein stability, and epistasis and quantitatively depicts the different costs inherent in the evolution of new functions.

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RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution

Cited by 19 publications

References 42 publications

Natural evolution of TEM-1 β-lactamase: experimental reconstruction and clinical relevance

Natural evolution of TEM-1 β-lactamase: experimental reconstruction and clinical relevance

Cell-free expression of RNA encoded genes using MS2 replicase

Shifting Fitness and Epistatic Landscapes Reflect Trade-offs along an Evolutionary Pathway

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