Integrons are genetic elements able to acquire and rearrange open reading frames (ORFs) embedded in gene cassette units and convert them to functional genes by ensuring their correct expression. They ...were originally identified as a mechanism used by Gram-negative bacteria to collect antibiotic resistance genes and express multiple resistance phenotypes in synergy with transposons. More recently, their role has been broadened with the discovery of chromosomal integron (CI) structures in the genomes of hundreds of bacterial species. This review focuses on the resources carried in these elements, on their unique recombination mechanisms, and on the different mechanisms controlling the cassette dynamics. We discuss the role of the toxin/antitoxin (TA) cassettes for the stabilization of the large cassette arrays carried in the larger CIs, known as superintegrons. Finally, we explore the central role played by single-stranded DNA in the integron cassette dynamics in light of the recent discovery that the integron integrase expression is controlled by the SOS response.
Bacterial biosensors, or bactosensors, are promising agents for medical and environmental diagnostics. However, the lack of scalable frameworks to systematically program ligand detection limits their ...applications. Here we show how novel, clinically relevant sensing modalities can be introduced into bactosensors in a modular fashion. To do so, we have leveraged a synthetic receptor platform, termed EMeRALD (Engineered Modularized Receptors Activated via Ligand-induced Dimerization) which supports the modular assembly of sensing modules onto a high-performance, generic signaling scaffold controlling gene expression in E. coli. We apply EMeRALD to detect bile salts, a biomarker of liver dysfunction, by repurposing sensing modules from enteropathogenic Vibrio species. We improve the sensitivity and lower the limit-of-detection of the sensing module by directed evolution. We then engineer a colorimetric bactosensor detecting pathological bile salt levels in serum from patients having undergone liver transplant, providing an output detectable by the naked-eye. The EMeRALD technology enables functional exploration of natural sensing modules and rapid engineering of synthetic receptors for diagnostics, environmental monitoring, and control of therapeutic microbes.
An inability to reliably predict quantitative behaviors for novel combinations of genetic elements limits the rational engineering of biological systems. We developed an expression cassette ...architecture for genetic elements controlling transcription and translation initiation in Escherichia coli: transcription elements encode a common mRNA start, and translation elements use an overlapping genetic motif found in many natural systems. We engineered libraries of constitutive and repressor-regulated promoters along with translation initiation elements following these definitions. We measured activity distributions for each library and selected elements that collectively resulted in expression across a 1,000-fold observed dynamic range. We studied all combinations of curated elements, demonstrating that arbitrary genes are reliably expressed to within twofold relative target expression windows with ∼93% reliability. We expect the genetic element definitions validated here can be collectively expanded to create collections of public-domain standard biological parts that support reliable forward engineering of gene expression at genome scales.
The inability to predict heterologous gene expression levels precisely hinders our ability to engineer biological systems. Using well-characterized regulatory elements offers a potential solution ...only if such elements behave predictably when combined. We synthesized 12,563 combinations of common promoters and ribosome binding sites and simultaneously measured DNA, RNA, and protein levels from the entire library. Using a simple model, we found that RNA and protein expression were within twofold of expected levels 80% and 64% of the time, respectively. The large dataset allowed quantitation of global effects, such as translation rate on mRNA stability and mRNA secondary structure on translation rate. However, the worst 5% of constructs deviated from prediction by 13-fold on average, which could hinder large-scale genetic engineering projects. The ease and scale this of approach indicates that rather than relying on prediction or standardization, we can screen synthetic libraries for desired behavior.
Synthetic biology often involves engineering microbial strains to express high-value proteins. Thanks to progress in rapid DNA synthesis and sequencing, deep learning has emerged as a promising ...approach to build sequence-to-expression models for strain optimization. But such models need large and costly training data that create steep entry barriers for many laboratories. Here we study the relation between accuracy and data efficiency in an atlas of machine learning models trained on datasets of varied size and sequence diversity. We show that deep learning can achieve good prediction accuracy with much smaller datasets than previously thought. We demonstrate that controlled sequence diversity leads to substantial gains in data efficiency and employed Explainable AI to show that convolutional neural networks can finely discriminate between input DNA sequences. Our results provide guidelines for designing genotype-phenotype screens that balance cost and quality of training data, thus helping promote the wider adoption of deep learning in the biotechnology sector.
SOS Response Controls Integron Recombination Guerin, Émilie; Cambray, Guillaume; Sanchez-Alberola, Neus ...
Science (American Association for the Advancement of Science),
05/2009, Letnik:
324, Številka:
5930
Journal Article
Recenzirano
Integrons are found in the genome of hundreds of environmental bacteria but are mainly known for their role in the capture and spread of antibiotic resistance determinants among Gram-negative ...pathogens. We report a direct link between this system and the ubiquitous SOS response. We found that LexA controlled expression of most integron integrases and consequently regulated cassette recombination. This regulatory coupling enhanced the potential for cassette swapping and capture in cells under stress, while minimizing cassette rearrangements or loss in constant environments. This finding exposes integrons as integrated adaptive systems and has implications for antibiotic treatment policies.
The practice of engineering biology now depends on the ad hoc reuse of genetic elements whose precise activities vary across changing contexts. Methods are lacking for researchers to affordably ...coordinate the quantification and analysis of part performance across varied environments, as needed to identify, evaluate and improve problematic part types. We developed an easy-to-use analysis of variance (ANOVA) framework for quantifying the performance of genetic elements. For proof of concept, we assembled and analyzed combinations of prokaryotic transcription and translation initiation elements in Escherichia coli. We determined how estimation of part activity relates to the number of unique element combinations tested, and we show how to estimate expected ensemble-wide part activity from just one or two measurements. We propose a new statistic, biomolecular part 'quality', for tracking quantitative variation in part performance across changing contexts.
Engineered bacteria are promising candidates for in situ detection and treatment of diseases. The female uro-genital tract presents several pathologies, such as sexually transmitted diseases or ...genital cancer, that could benefit from such technology. While bacteria from the gut microbiome are increasingly engineered, the use of chassis isolated from the female uro-genital resident flora has been limited. A major hurdle to implement the experimental throughput required for efficient engineering in these non-model bacteria is their low transformability. Here we report an optimized electrotransformation protocol for Lactobacillus jensenii, one the most widespread species across vaginal microflora. Starting from classical conditions, we optimized buffers, electric field parameters, cuvette type and DNA quantity to achieve an 80-fold improvement in transformation efficiency, with up to 3.5·103 CFUs/μg of DNA in L. jensenii ATCC 25258. We also identify several plasmids that are maintained and support reporter gene expression in L. jensenii. Finally, we demonstrate that our protocol provides increased transformability in three independent clinical isolates of L. jensenii. This work will facilitate the genetic engineering of L. jensenii and enable its use for addressing challenges in gynecological healthcare.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Chromosome 2 of Vibrio cholerae carries a chromosomal superintegron, composed of an integrase, a cassette integration site (attI) and an array of mostly promoterless gene cassettes. We determined the ...precise location of the promoter, Pc, which drives the transcription of the first cassettes of the V. cholerae superintegron. We found that cassette mRNA starts 65 bp upstream of the attI site, so that the inversely oriented promoters Pc and Pint (integrase promoter) partly overlap, allowing for their potential co-regulation. Pint was previously shown to be induced during the SOS response and is further controlled by the catabolite repression cAMP-CRP complex. We found that cassette expression from Pc was also controlled by the cAMP-CRP complex, but is not part of the SOS regulon. Pint and Pc promoters were both found to be induced in rich medium, at high temperature, high salinity and at the end of exponential growth phase, although at very different levels and independently of sigma factor RpoS. All these results show that expression from the integrase and cassette promoters can take place at the same time, thus leading to coordinated excisions and integrations within the superintegron and potentially coupling cassette shuffling to immediate selective advantage.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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