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Proteins that are kinetically stable are thought to be less prone to both aggregation and proteolysis. We demonstrate that the classical lac system of Escherichia coli can be leveraged as a model system to study this relation. β-galactosidase (LacZ) plays a critical role in lactose metabolism and is an extremely stable protein that can persist in growing cells for multiple generations after expression has stopped. By attaching degradation tags to the LacZ protein, we find that LacZ can be transiently degraded during lac operon expression but once expression has stopped functional LacZ is protected from degradation. We reversibly destabilize its tetrameric assembly using α-complementation, and show that unassembled LacZ monomers and dimers can either be degraded or lead to formation of aggregates within cells, while the tetrameric state protects against proteolysis and aggregation. We show that the presence of aggregates is associated with cell death, and that these proteotoxic stress phenotypes can be alleviated by attaching an ssrA tag to LacZ monomers which leads to their degradation. We unify our findings using a biophysical model that enables the interplay of protein assembly, degradation, and aggregation to be studied quantitatively in vivo. This work may yield approaches to reversing and preventing protein-misfolding disease states, while elucidating the functions of proteolytic stability in constant and fluctuating environments. [ABSTRACT FROM AUTHOR]

ABSTRACTMany undergraduates struggle to interpret abstract concepts in molecular biology. Modeling can facilitate learning by making these abstract concepts tangible. Here, we present an exercise based on the lac operon designed for undergraduate students using LEGO bricks. The lac operon is a classic example of transcriptional regulation taught in a variety of undergraduate biology courses and is fundamental to understanding the regulation of gene expression. This easy-to-implement active learning exercise demonstrates how the various components of the lac operon are oriented under a variety of nutritional conditions to control gene expression. In addition, higher-order concepts, such as the effect of mutation on lac operon expression, can be readily modeled. Overall, students not only found this exercise to be enjoyable but also helpful as a tool to engage with this course material.

DNA looping has emerged as a central paradigm of transcriptional regulation, as it is shared across many living systems. One core property of DNA looping-based regulation is its ability to greatly enhance repression or activation of genes with only a few copies of transcriptional regulators. However, this property based on a small number of proteins raises the question of the robustness of such a mechanism with respect to the large intracellular perturbations taking place during growth and division of the cell. Here we address the issue of sensitivity to variations of intracellular parameters of gene regulation by DNA looping. We use the lac system as a prototype to experimentally identify the key features of the robustness of DNA looping in growing Escherichia coli cells. Surprisingly, we observe time intervals of tight repression spanning across division events, which can sometimes exceed 10 generations. Remarkably, the distribution of such long time intervals exhibits memoryless statistics that is mostly insensitive to repressor concentration, cell division events, and the number of distinct loops accessible to the system. By contrast, gene regulation becomes highly sensitive to these perturbations when DNA looping is absent. Using stochastic simulations, we propose that the observed robustness to division emerges from the competition between fast, multiple rebinding events of repressors and slow initiation rate of the RNA polymerase. We argue that fast rebinding events are a direct consequence of DNA looping that ensures robust gene repression across a range of intracellular perturbations.

We present here a thorough and complete analysis of mouse P0-P140 prethalamic histogenetic subdivisions and corresponding nuclear derivatives, in the context of local tract landmarks. The study used as fundamental material brains from a transgenic mouse line that expresses LacZ under the control of an intragenic enhancer of Dlx5 and Dlx6 (Dlx5/6-LacZ). Subtle shadings of LacZ signal, jointly with pan-DLX immunoreaction, and several other ancillary protein or RNA markers, including Calb2 and Nkx2.2 ISH (for the prethalamic eminence, and derivatives of the rostral zona limitans shell domain, respectively) were mapped across the prethalamus. The resulting model of the prethalamic region postulates tetrapartite rostrocaudal and dorsoventral subdivisions, as well as a tripartite radial stratification, each cell population showing a characteristic molecular profile. Some novel nuclei are proposed, and some instances of potential tangential cell migration were noted.

The Escherichia coli system of Cairns and Foster employs a lac frameshift mutation that reverts rarely (10-9/cell/division) during unrestricted growth. However, when 108 cells are plated on lactose medium, the nongrowing lawn produces ∼50 Lac+ revertant colonies that accumulate linearly with time over 5 days. Revertants carry very few associated mutations. This behavior has been attributed to an evolved mechanism ("adaptive mutation" or "stress-induced mutagenesis") that responds to starvation by preferentially creating mutations that improve growth. We describe an alternative model, "selective inbreeding," in which natural selection acts during intercellular transfer of the plasmid that carries the mutant lac allele and the dinB gene for an error-prone polymerase. Revertant genome sequences show that the plasmid is more intensely mutagenized than the chromosome. Revertants vary widely in their number of plasmid and chromosomal mutations. Plasmid mutations are distributed evenly, but chromosomal mutations are focused near the replication origin. Rare, heavily mutagenized, revertants have acquired a plasmid tra mutation that eliminates conjugation ability. These findings support the new model, in which revertants are initiated by rare pre-existing cells (105) with many copies of the F'lac plasmid. These cells divide under selection, producing daughters that mate. Recombination between donor and recipient plasmids initiates rolling-circle plasmid over-replication, causing a mutagenic elevation of DinB level. A lac+ reversion event starts chromosome replication and mutagenesis by accumulated DinB. After reversion, plasmid transfer moves the revertant lac+ allele into an unmutagenized cell, and away from associated mutations. Thus, natural selection explains why mutagenesis appears stress-induced and directed.

Tight coordination between transcription and translation is crucial to maintaining the integrity of gene expression in bacteria, yet how bacteria manage to coordinate these two processes remains unclear. Possible direct physical coupling between the RNA polymerase and ribosome has been thoroughly investigated in recent years. Here, we quantitatively characterize the transcriptional kinetics of Escherichia coli under different growth conditions. Transcriptional and translational elongation remain coordinated under various nutrient conditions, as previously reported. However, transcriptional elongation was not affected under antibiotics that slowed down translational elongation. This result was also found by introducing nonsense mutation that completely dissociated transcription from translation. Our data thus provide direct evidence that translation is not required to maintain the speed of transcriptional elongation. In cases where transcription and translation are dissociated, our study provides quantitative characterization of the resulting process of premature transcriptional termination (PTT). PTT-mediated polarity caused by translation-targeting antibiotics substantially affected the coordinated expression of genes in several long operons, contributing to the key physiological effects of these antibiotics. Our results also suggest a model in which the coordination between transcriptional and translational elongation under normal growth conditions is implemented by guanosine tetraphosphate.

The lac operon in E. coli has been extensively studied by computational biologists. The bacterium uses it to survive in the absence of glucose, utilizing lactose for growth. This paper presents a novel modeling mechanism for the lac operon, transferring the process of lactose metabolism from the cell to a finite state machine (FSM). This FSM is implemented in field-programmable gate array (FPGA) and simulations are run in random conditions. A Markov chain is also proposed for the lac operon, which helps study its behavior in terms of probabilistic variables, validating the finite state machine at the same time. This work is focused towards conversion of biological processes into computing machines.

Since Jacob and Monod’s discovery of the lac operon ∼1960, the explanations offered for most metabolic adaptations have been genetic. The focus has been on the adaptive changes in gene expression that occur, which are often referred to as “metabolic reprogramming.” The contributions metabolism makes to adaptation have been largely ignored. Here we point out that metabolic adaptations, including the associated changes in gene expression, are highly dependent on the metabolic state of an organism prior to the environmental change to which it is adapting, and on the plasticity of that state. In support of this hypothesis, we examine the paradigmatic example of a genetically driven adaptation, the adaptation of E. coli to growth on lactose, and the paradigmatic example of a metabolic driven adaptation, the Crabtree effect in yeast. Using a framework based on metabolic control analysis, we have reevaluated what is known about both adaptations, and conclude that knowledge of the metabolic properties of these organisms prior to environmental change is critical for understanding not only how they survive long enough to adapt, but also how the ensuing changes in gene expression occur, and their phenotypes post-adaptation. It would be useful if future explanations for metabolic adaptations acknowledged the contributions made to them by metabolism, and described the complex interplay between metabolic systems and genetic systems that make these adaptations possible.

Polycyclic aromatic hydrocarbons like benzo[a]pyrene (BaP) are ubiquitous environmental contaminants formed during incomplete combustion of organic materials. Our prior work showed that transplacental exposure to BaP depletes ovarian follicles and increases prevalence of epithelial ovarian tumors later in life. We used the MutaMouse transgenic rodent model to address the hypothesis that ovarian mutations play a role in tumorigenesis caused by prenatal exposure to BaP. Pregnant MutaMouse females were treated with 0, 10, 20, or 40 mg/(kg day) BaP orally on gestational days 7-16, covering critical windows of ovarian development. Female offspring were euthanized at 10 weeks of age; some ovaries with oviducts were processed for follicle counting; other ovaries/oviducts and bone marrow were processed for determination of lacZ mutant frequency (MF). Mutant plaques were pooled within dose groups and sequenced to determine the mutation spectrum. BaP exposure caused highly significant dose-related decreases in ovarian follicles and increases in ovarian/oviductal and bone marrow mutant frequencies at all doses. Absence of follicles, cell packets, and epithelial tubular structures were observed with 20 and 40 mg/(kg day) BaP. Depletion of ovarian germ cells was inversely associated with ovarian MF. BaP induced primarily G > T and G > C transversions and deletions in ovaries/oviducts and bone marrow cells and produced a mutation signature highly consistent with that of tobacco smoking in human cancers. Overall, our results show that prenatal BaP exposure significantly depletes ovarian germ cells, causes histopathological abnormalities, and increases the burden of ovarian/oviductal mutations, which may be involved in pathogenesis of epithelial ovarian tumors. Environ. Mol. Mutagen. 60:410-420, 2019. © 2018 Her Majesty the Queen in Right of Canada.

Lacticaseibacillus casei is used extensively in the fermented milk-beverage industry as a starter culture. Acid production capacity during fermentation is the main criterion for evaluating starters although it is strain-dependent. In this study, the acid production rates of 114 L. casei strains were determined and then classified into high acid (HC), medium acid (MC), and low acid (LC) groups. Comparative genomics analysis found that the lac operon genes encoding the phosphoenolpyruvate-lactose phosphotransferase system (PTSLac) were located on plasmids in the HC strains; however, it is notable that the corresponding operons were located on the chromosome in LC strains. Real-time PCR analysis showed that the copy numbers of lac operon genes in HC strains were between 3.1 and 9.3. To investigate the relationship between copy number and acid production rate, the lac operon cluster of the HC group was constitutively expressed in LC strains. The resulting copy numbers of lac operon genes were between 15.8 and 18.1; phospho-β-galactosidase activity increased by 1.68–1.99-fold; and the acid production rates increased by 1.24–1.40-fold, which enhanced the utilization rate of lactose from 17.5 to 42.6% in the recombinant strains. The markedly increased expression of lac operon genes increased lactose catabolism and thereby increased the acid production rate of L. casei. [ABSTRACT FROM AUTHOR]

The lac operon in E. coli has been extensively studied by computational biologists. The bacterium uses it to survive in the absence of glucose, utilizing lactose for growth. This paper presents a novel modeling mechanism for the lac operon, transferring the process of lactose metabolism from the cell to a finite state machine (FSM). This FSM is implemented in field-programmable gate array (FPGA) and simulations are run in random conditions. A Markov chain is also proposed for the lac operon, which helps study its behavior in terms of probabilistic variables, validating the finite state machine at the same time. This work is focused towards conversion of biological processes into computing machines. [ABSTRACT FROM AUTHOR]

To test whether growth limitation induces mutations, Cairns and Foster constructed an Escherichia coli strain whose mutant lac allele provides 1-2% of normal ability to use lactose. This strain cannot grow on lactose, but produces ∼50 Lac+ revertant colonies per 108 plated cells over 5 days. About 80% of revertants carry a stable lac+ mutation made by the error-prone DinB polymerase, which may be induced during growth limitation; 10% of Lac+ revertants are stable but form without DinB; and the remaining 10% grow by amplifying their mutant lac allele and are unstably Lac+ Induced DinB mutagenesis has been explained in two ways: (1) upregulation of dinB expression in nongrowing cells ("stress-induced mutagenesis") or (2) selected local overreplication of the lac and dinB+ genes on lactose medium (selected amplification) in cells that are not dividing. Transcription of dinB is necessary but not sufficient for mutagenesis. Evidence is presented that DinB enhances reversion only when encoded somewhere on the F'lac plasmid that carries the mutant lac gene. A new model will propose that rare preexisting cells (1 in a 1000) have ∼10 copies of the F'lac plasmid, providing them with enough energy to divide, mate, and overreplicate their F'lac plasmid under selective conditions. In these clones, repeated replication of F'lac in nondividing cells directs opportunities for lac reversion and increases the copy number of the dinB+ gene. Amplification of dinB+ increases the error rate of replication and increases the number of lac+ revertants. Thus, reversion is enhanced in nondividing cells not by stress-induced mutagenesis, but by selected coamplification of the dinB and lac genes, both of which happen to lie on the F'lac plasmid.

Here, we describe a free, web‐based simulation of the lac operon, "LacOp," that is designed to enhance the learning of prokaryotic gene regulation and pathways in advanced high school and undergraduate genetics courses. This new electronic resource was created by a team of students in an advanced undergraduate course and is hosted online (http://flask-env.rnwhymamqf.us-west-2.elasticbeanstalk.com/lacop). LacOp has a simple web interface compatible with a range of devices, including smartphones. To determine whether the LacOp simulation enhances student learning from traditional instruction, we introduced the lac operon to undergraduate genetics students through a traditional classroom experience followed by use of the LacOp simulation. Students worked on their own using the included tutorial to create and test the effect of various genotypes on E. coli lactose metabolism and regulation. Upon completion of the tutorial, students showed measurable gains in conceptual understanding of the lac operon. These students also reported a generally favorable opinion of the LacOP simulation as a use of their instructional time. [ABSTRACT FROM AUTHOR]

Computational models of enhancer function generally assume that transcription factors (TFs) exert their regulatory effects independently, modeling an enhancer as a "bag of sites." These models fail on endogenous loci that harbor multiple enhancers, and a "two-tier" model appears better suited: in each enhancer TFs work independently, and the total expression is a weighted sum of their expression readouts. Here, we test these two opposing views on how cis-regulatory information is integrated. We fused two Drosophila blastoderm enhancers, measured their readouts, and applied the above two models to these data. The two-tier mechanism better fits these readouts, suggesting that these fused enhancers comprise multiple independent modules, despite having sequence characteristics typical of single enhancers. We show that short-range TF-TF interactions are not sufficient to designate such modules, suggesting unknown underlying mechanisms. Our results underscore that mechanisms of how modules are defined and how their outputs are combined remain to be elucidated.

Natural prokaryotic gene repression systems often exploit DNA looping to increase the local concentration of gene repressor proteins at a regulated promoter via contributions from repressor proteins bound at distant sites. Using principles from the Escherichia coli lac operon we design analogous repression systems based on target sequence-programmable Transcription Activator-Like Effector dimer (TALED) proteins. Such engineered switches may be valuable for synthetic biology and therapeutic applications. Previous TALEDs with inducible non-covalent dimerization showed detectable, but limited, DNA loop-based repression due to the repressor protein dimerization equilibrium. Here, we show robust DNA loop-dependent bacterial promoter repression by covalent TALEDs and verify that DNA looping dramatically enhances promoter repression in E. coli. We characterize repression using a thermodynamic model that quantitates this favorable contribution of DNA looping. This analysis unequivocally and quantitatively demonstrates that optimized TALED proteins can drive loop-dependent promoter repression in E. coli comparable to the natural LacI repressor system. This work elucidates key design principles that set the stage for wide application of TALED-dependent DNA loop-based repression of target genes., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

pET vectors allow inducible expression of recombinant proteins in Escherichia coli. In this system, isopropyl β-d-1-thiogalactopyranoside (IPTG) drives lacUV5 promoter to produce T7 RNA polymerase, simultaneously releasing the suppression of T7lac promoter. T7 RNA polymerase then strongly transcribes the target gene. A lac repressor encoded by lacI in the vector represses the promoters. Despite stringent repression and inducible expression achieved with the pET system, unexpected leaky expression can occur without IPTG induction. Here, by evaluating leaky expression in recombinant cells cultured in various Luria-Bertani (LB) media, prepared using yeast extract and peptone from different suppliers, as well as in five commercial premix-LB media, we confirmed the presence of unknown lac inducers in LB. To explore these inducers, we examined E. coli growth in media comprising yeast extract or peptone. At 4% concentration, five commercial yeast extract and six peptone samples individually allowed E. coli growth equivalent to that in LB medium. We determined the luciferase activity of the luxCDABE operon in the pET vector under these conditions. The presence of different concentrations of inducers was detected in both the yeast extract and peptone. Furthermore, we blended yeast extract and peptone with low or high concentrations of lac inducers. The low-expression blend, used as a basal medium before IPTG addition, allowed leak-free, tightly controlled expression. The high-expression blend was used for constitutive high-expression and pET induction with the basal medium, in lieu of IPTG. These blended media can be used for well-controlled inducible and constitutive expression using the pET system., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

As temperature serves as a versatile input signal, thermoresponsive genetic controls have gained significant interest for recombinant protein production and metabolic engineering applications. The conventional thermoresponsive systems normally require the continuous exposure of heat stimuli to trigger the prolonged expression of targeted genes, and the accompanied heat-shock response is detrimental to the bioproduction process. In this study, we present the design of thermoresponsive quorum-sensing (ThermoQS) circuits to make Escherichia coli record transient heat stimuli. By conversion of the heat input into the accumulation of quorum-sensing molecules such as acyl-homoserine lactone derived from Pseudomonas aeruginosa , sustained gene expressions were achieved by a minimal heat stimulus. Moreover, we also demonstrated that we reprogrammed the E. coli Lac operon to make it respond to heat stimuli with an impressive signal-to-noise ratio (S/N) of 15.3. Taken together, we envision that the ThermoQS systems reported in this study are expected to remarkably diminish both design and experimental expenditures for future metabolic engineering applications.

Optogenetics' advancement has made light induction attractive for controlling biological processes due to its advantages of fine-tunability, reversibility, and low toxicity. The lactose operon induction system, commonly used in Escherichia coli, relies on the binding of lactose or isopropyl β-d-1-thiogalactopyranoside (IPTG) to the lactose repressor protein LacI, playing a pivotal role in controlling the lactose operon. Here, we harnessed the light-responsive light-oxygen-voltage 2 (LOV2) domain from Avena sativa phototropin 1 as a tool for light control and engineered LacI into two light-responsive variants, OptoLacIL and OptoLacID. These variants exhibit direct responsiveness to light and darkness, respectively, eliminating the need for IPTG. Building upon OptoLacI, we constructed two light-controlled E. coli gene expression systems, OptoE.coliLight system and OptoE.coliDark system. These systems enable bifunctional gene expression regulation in E. coli through light manipulation and show superior controllability compared to IPTG-induced systems. We applied the OptoE.coliDark system to protein production and metabolic flux control. Protein production levels are comparable to those induced by IPTG. Notably, the titers of dark-induced production of 1,3-propanediol (1,3-PDO) and ergothioneine exceeded 110% and 60% of those induced by IPTG, respectively. The development of OptoLacI will contribute to the advancement of the field of optogenetic protein engineering, holding substantial potential applications across various fields., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Systems like the prototypical lac operon can reliably hold repression of transcription upon DNA replication across cell cycles with just 10 repressor molecules per cell and behave as if they were at equilibrium. The origin of this phenomenology is still an unresolved question. Here, we develop a general theory to analyze strong perturbations in quasi-equilibrium systems and use it to quantify the effects of DNA replication in gene regulation. We find a scaling law linking actual with predicted equilibrium transcription via a single kinetic parameter. We show that even the lac operon functions beyond the physical limits of naive regulation through compensatory mechanisms that suppress non-equilibrium effects. Synthetic systems without adjuvant activators, such as the cAMP receptor protein (CRP), lack this reliability. Our results provide a rationale for the function of CRP, beyond just being a tunable activator, as a mitigator of cell cycle perturbations., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Lactic acid (lactate) is among the most important natural α-hydroxy acids and is involved in a number of biological processes. Lactate is the terminal product of glucose metabolism in tissues under oxygen limitation, the major intercellular energy compound in human muscles and brain tissue, and a natural component of wines and other foodstuffs. Many microorganisms utilize L-lactate as a carbon and energy source, which provides for their survival and competitiveness with other species. Ability to utilize L-lactate is responsible for pathogenicity of some bacterial species, which is important in medical and pharmaceutical research. L-lactate-oxidizing enzymes are subdivided into soluble and insoluble ones. Soluble L-lactate oxidase systems include flavin-containing enzymes, which transfer electrons directly to oxygen (L-lactate oxidases and L-lactate monooxygenases). They are common among bacteria, yeasts, and fungi. Insoluble enzymes may contain both flavins and other components, including Fe-S clusters, which transfer electrons to the respiratory chain at the level of ubiquinone (coenzyme Q or menaquinone). In this case lactate oxidation is coupled to development of the transmembrane potential and ATP synthesis. These systems are present mostly in bacteria and algae. Investigation of the structure and organization of L-lactate utilization operons is an important aspect in investigation of L-lactate oxidase systems, since this approach may result in identification of new enzymes involved in lactate oxidation. [ABSTRACT FROM AUTHOR]

Chromosomal integration offers a selection-free alternative to DNA plasmids for expression of foreign proteins and metabolic pathways. Episomal plasmid DNA is convenient but has drawbacks including increased metabolic burden and the requirement for selection in the form of antibiotics. E. coli has long been used for the expression of foreign proteins and for the production of valuable metabolites by expression of complete metabolic pathways. The gene encoding the fluorescent reporter protein mCherry was integrated into four genomic loci on the E. coli chromosome to measure protein expression at each site. Expression levels ranged from 25% to 500% compared to the gene expressed on a high-copy plasmid. Modular expression of DNA is one of the most commonly used methods for optimizing metabolite production by metabolic engineering. By combining a recently developed method for integration of large synthetic DNA constructs into the genome, we were able to integrate two foreign pathways into the same four genomic loci. We have demonstrated that only one of the genomic loci resulted in the production of violacein, and that all four loci produced trans-cinnamic acid from the TAL pathway.

DNA double-strand breaks (DSBs) leading to loss of nucleotides in the transcribed region can be lethal. Classical non-homologous end-joining (C-NHEJ) is the dominant pathway for DSB repair (DSBR) in adult mammalian cells. Here we report that during such DSBR, mammalian C-NHEJ proteins form a multiprotein complex with RNA polymerase II and preferentially associate with the transcribed genes after DSB induction. Depletion of C-NHEJ factors significantly abrogates DSBR in transcribed but not in non-transcribed genes. We hypothesized that nascent RNA can serve as a template for restoring the missing sequences, thus allowing error-free DSBR. We indeed found pre-mRNA in the C-NHEJ complex. Finally, when a DSB-containing plasmid with several nucleotides deleted within the E. coli lacZ gene was allowed time to repair in lacZ-expressing mammalian cells, a functional lacZ plasmid could be recovered from control but not C-NHEJ factor-depleted cells, providing important mechanistic insights into C-NHEJ-mediated error-free DSBR of the transcribed genome.

The main tenet of physical biology is that biological phenomena can be subject to the same quantitative and predictive understanding that physics has afforded in the context of inanimate matter. However, the inherent complexity of many of these biological processes often leads to the derivation of complex theoretical descriptions containing a plethora of unknown parameters. Such complex descriptions pose a conceptual challenge to the establishment of a solid basis for predictive biology. In this article, we present various exciting examples of how synthetic biology can be used to simplify biological systems and distill these phenomena down to their essential features as a means to enable their theoretical description. Here, synthetic biology goes beyond previous efforts to engineer nature and becomes a tool to bend nature to understand it. We discuss various recent and classic experiments featuring applications of this synthetic approach to the elucidation of problems ranging from bacteriophage infection, to transcriptional regulation in bacteria and in developing embryos, to evolution. In all of these examples, synthetic biology provides the opportunity to turn cells into the equivalent of a test tube, where biological phenomena can be reconstituted and our theoretical understanding put to test with the same ease that these same phenomena can be studied in the in vitro setting.

Live cell RNA imaging using genetically encoded fluorescent labels is an important tool for monitoring RNA activities. A recently reported RNA aptamer-fluorogen system, the Spinach, in which an RNA aptamer binds and induces the fluorescence of a GFP-like 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) ligand, can be readily tagged to the RNA of interest. Although the aptamer-fluorogen system is sufficient for imaging highly abundant non-coding RNAs (tRNAs, rRNAs, etc.), it performs poorly for mRNA imaging due to low brightness. In addition, whether the aptamer-fluorogen system may perturb the native RNA characteristics has not been systematically characterized at the levels of RNA transcription, translation and degradation. To increase the brightness of these aptamer-fluorogen systems, we constructed and tested tandem arrays containing multiple Spinach aptamers (8-64 aptamer repeats). Such arrays enhanced the brightness of the tagged mRNA molecules by up to ~17 fold in living cells. Strong laser excitation with pulsed illumination further increased the imaging sensitivity of Spinach array-tagged RNAs. Moreover, transcriptional fusion to the Spinach array did not affect mRNA transcription, translation or degradation, indicating that aptamer arrays might be a generalizable labeling method for high-performance and low-perturbation live cell RNA imaging.

Our previous study identified a new β-galactosidase in Erwinia sp. E602. To further understand the lactose metabolism in this strain, de novo genome assembly was conducted by using a strategy combining Illumina and PacBio sequencing technology. The whole genome of Erwinia sp. E602 includes a 4.8 Mb chromosome and a 326 kb large plasmid. A total of 4,739 genes, including 4,543 protein-coding genes, 25 rRNAs, 82 tRNAs and 7 other ncRNAs genes were annotated. The plasmid was the largest one characterized in genus Erwinia by far, and it contained a number of genes and pathways responsible for lactose metabolism and regulation. Moreover, a new plasmid-borne lac operon that lacked a typical β-galactoside transacetylase (lacA) gene was identified in the strain. Phylogenetic analysis showed that the genes lacY and lacZ in the operon were under positive selection, indicating the adaptation of lactose metabolism to the environment in Erwinia sp. E602. Our current study demonstrated that the hybrid de novo genome assembly using Illumina and PacBio sequencing technologies, as well as the metabolic pathway analysis, provided a useful strategy for better understanding of the evolution of undiscovered microbial species or strains. [ABSTRACT FROM AUTHOR]

Our previous study identified a new β-galactosidase in Erwinia sp. E602. To further understand the lactose metabolism in this strain, de novo genome assembly was conducted by using a strategy combining Illumina and PacBio sequencing technology. The whole genome of Erwinia sp. E602 includes a 4.8 Mb chromosome and a 326 kb large plasmid. A total of 4,739 genes, including 4,543 protein-coding genes, 25 rRNAs, 82 tRNAs and 7 other ncRNAs genes were annotated. The plasmid was the largest one characterized in genus Erwinia by far, and it contained a number of genes and pathways responsible for lactose metabolism and regulation. Moreover, a new plasmid-borne lac operon that lacked a typical β-galactoside transacetylase (lacA) gene was identified in the strain. Phylogenetic analysis showed that the genes lacY and lacZ in the operon were under positive selection, indicating the adaptation of lactose metabolism to the environment in Erwinia sp. E602. Our current study demonstrated that the hybrid de novo genome assembly using Illumina and PacBio sequencing technologies, as well as the metabolic pathway analysis, provided a useful strategy for better understanding of the evolution of undiscovered microbial species or strains.

Many undergraduates struggle to interpret abstract concepts in molecular biology. Modeling can facilitate learning by making these abstract concepts tangible. Here, we present an exercise based on the lac operon designed for undergraduate students using LEGO bricks. The lac operon is a classic example of transcriptional regulation taught in a variety of undergraduate biology courses and is fundamental to understanding the regulation of gene expression. This easy-to-implement active learning exercise demonstrates how the various components of the lac operon are oriented under a variety of nutritional conditions to control gene expression. In addition, higher-order concepts, such as the effect of mutation on lac operon expression, can be readily modeled. Overall, students not only found this exercise to be enjoyable but also helpful as a tool to engage with this course material., Competing Interests: The authors declare no conflict of interest.

Pasteurella multocida is a zoonotic conditional pathogen that infects multiple livestock species, causing substantial economic losses in the animal husbandry industry. An efficient markerless method for gene manipulation may facilitate the investigations of P. multocida gene function and pathogenesis of P. multocida . Herein, a temperature-sensitive shuttle vector was constructed using lacZ as a selection marker, and markerless glgB , opa , and hyaE mutants of P. multocida were subsequently constructed through blue-white colony screening. The screening efficiency of markerless deletion strains was improved by the lacZ system, and the method could be used for multiple gene deletions. However, the fur mutant was unavailable via this method. Therefore, we constructed a pheS m screening system based on mutated phenylalanine tRNA synthetase as a counterselection marker to achieve fur deletion mutant. The transformed strain was sensitive to 20 mM p -chloro-phenylalanine, demonstrating the feasibility of pheS m as a counter-selective marker. The pheS m system was used for markerless deletions of glgB , opa , and hyaE as well as fur that could not be screened by the lacZ system. A comparison of screening efficiencies of the system showed that the pheS m counterselection system was more efficient than the lacZ system and broadly applicable for mutant screening. The methods developed herein may provide valuable tools for genetic manipulation of P. multocida .IMPORTANCE Pasteurella multocida is a highly contagious zoonotic pathogen. An understanding of its underlying pathogenic mechanisms is of considerable importance and requires efficient species-specific genetic tools. Herein, we propose a screening system for P. multocida mutants using lacZ or pheS m screening markers. We evaluated the efficiencies of both systems, which were used to achieve markerless deletion of multiple genes. The results of this study support the use of lacZ or pheS m as counterselection markers to improve counterselection efficiency in P. multocida . This study provides an effective genetic tool for investigations of the virulence gene functions and pathogenic mechanisms of P. multocida ., Competing Interests: The authors declare no conflict of interest.

The lac operon is one of the best known gene regulatory circuits and constitutes a landmark example of how bacteria tune their metabolism to nutritional conditions. It is nearly ubiquitous in Escherichia coli strains justifying the use of its phenotype, the ability to consume lactose, for species identification. Lactose is the primary sugar found in milk, which is abundant in mammals during the first weeks of life. However, lactose is virtually non-existent after the weaning period, with humans being an exception as many consume dairy products throughout their lives. The absence of lactose during adulthood in most mammals and the rarity of lactose in the environment, means that the selective pressure for maintaining the lac operon could be weak for long periods of time. Despite the ability to metabolize lactose being a hallmark of E. coli 's success when colonizing its primary habitat, the mammalian intestine, the selective value of this trait remains unknown in this ecosystem during adulthood. Here we determine the competitive advantage conferred by the lac operon to a commensal strain of E. coli when colonizing the mouse gut. We find that its benefit, which can be as high as 11%, is contingent on the presence of lactose in the diet and on the presence of other microbiota members in the gut, but the operon is never deleterious. These results help explaining the pervasiveness of the lac operon in E. coli , but also its polymorphism, as lac -negative E. coli strains albeit rare can naturally occur in the gut. [ABSTRACT FROM AUTHOR]

Mechanistic computational models enable the study of regulatory mechanisms implicated in various biological processes. These models provide a means to analyze the dynamics of the systems they describe, and to study and interrogate their properties, and provide insights about the emerging behavior of the system in the presence of single or combined perturbations. Aimed at those who are new to computational modeling, we present here a practical hands-on protocol breaking down the process of mechanistic modeling of biological systems in a succession of precise steps. The protocol provides a framework that includes defining the model scope, choosing validation criteria, selecting the appropriate modeling approach, constructing a model and simulating the model. To ensure broad accessibility of the protocol, we use a logical modeling framework, which presents a lower mathematical barrier of entry, and two easy-to-use and popular modeling software tools: Cell Collective and GINsim. The complete modeling workflow is applied to a well-studied and familiar biological process—the lac operon regulatory system. The protocol can be completed by users with little to no prior computational modeling experience approximately within 3 h. [ABSTRACT FROM AUTHOR]

Populations of Escherichia coli selected in constant and fluctuating environments containing lactose often adapt by substituting mutations in the lacI repressor that cause constitutive expression of the lac operon. These mutations occur at a high rate and provide a significant benefit. Despite this, eight of 24 populations evolved for 8,000 generations in environments containing lactose contained no detectable repressor mutations. We report here on the basis of this observation. We find that, given relevant mutation rates, repressor mutations are expected to have fixed in all evolved populations if they had maintained the same fitness effect they confer when introduced to the ancestor. In fact, reconstruction experiments demonstrate that repressor mutations have become neutral or deleterious in those populations in which they were not detectable. Populations not fixing repressor mutations nevertheless reached the same fitness as those that did fix them, indicating that they followed an alternative evolutionary path that made redundant the potential benefit of the repressor mutation, but involved unique mutations of equivalent benefit. We identify a mutation occurring in the promoter region of the uspB gene as a candidate for influencing the selective choice between these paths. Our results detail an example of historical contingency leading to divergent evolutionary outcomes. [ABSTRACT FROM AUTHOR]

Bioengineered bladder tissue is needed for patients with neurogenic bladder disease as well as for cancer. Current technologies in bladder tissue engineering have been hampered by an inability to efficiently initiate blood supply to the graft, ultimately leading to complications that include graft contraction, ischemia, and perforation. To date, the biological mechanisms of vascularization on transplant have not been suitably investigated for urologic tissues. To better understand the mechanisms of neovascularization on bladder wall transplant, a chimeric mouse model was generated such that angiogenesis and vasculogenesis could be independently assessed in vivo. Green fluorescence protein (GFP) transgenic mice received bone marrow transplants from β-galactosidase (LacZ) transgenic animals and then subsequent bladder wall transplants from wild-type donor mice. Before euthanization, the aorta was infused with fluorescent microbeads (fluorospheres) to identify perfused vessels. The contributions of GFP (angiogenesis) and LacZ (vasculogenesis) to the formation of CD31-expressing blood vessels within the wild-type graft were evaluated by immunohistochemistry at different time points and locations within the graft (proximal, middle, and distal) to provide a spatiotemporal analysis of neovascularization. The GFP index, a measure of angiogenic host ingrowth, was significantly higher at proximal versus mid or distal regions in animals 2-16 weeks post-transplant. However, GFP index did not increase over time in any area. Within 7 days post-transplant, perfusion of primarily wild-type, donor blood vessels in the most distal areas of the graft was observed by intraluminal fluorospheres. In addition, chimeric host-donor (GFP-wild type) blood vessels were evident in proximal areas. The contribution of vasculogenesis to vascularization of the graft was limited, as LacZ cells were not specifically associated with the endothelial cells of blood vessels, but rather found primarily in areas of inflammation. The data suggest that angiogenesis of host blood vessels into the proximal region leads to inosculation between host and donor vessels and subsequent perfusion of the graft via pre-existing graft vessels within the first week after transplant. As such, the engineering of graft blood vessels and the promotion of inosculation might prevent graft contraction, thereby potentiating the use of bioengineered bladder tissue for transplantation.

Neural crest cells (NCC) comprise a multipotent, migratory stem cell and progenitor population that gives rise to numerous cell and tissue types within a developing embryo, including craniofacial bone and cartilage, neurons and glia of the peripheral nervous system, and melanocytes within the skin. Here we describe two novel stable transgenic mouse lines suitable for lineage tracing and analysis of gene function in NCC. Firstly, using the F10N enhancer of the Mef2c gene (Mef2c-F10N) linked to LacZ, we generated transgenic mice (Mef2c-F10N-LacZ) that express LacZ in the majority, if not all migrating NCC that delaminate from the neural tube. Mef2c-F10N-LacZ then continues to be expressed primarily in neurogenic, gliogenic and melanocytic NCC and their derivatives, but not in ectomesenchymal derivatives. Secondly, we used the same Mef2c-F10N enhancer together with Cre recombinase to generate transgenic mice (Mef2c-F10N-Cre) that can be used to indelibly label, or alter gene function in, migrating NCC and their derivatives. At early stages of development, Mef2c-F10N-LacZ and Mef2c-F10N-Cre label NCC in a pattern similar to Wnt1-Cre mice, with the exception that Mef2c-F10N-LacZ and Mef2c-F10N-Cre specifically label NCC that have delaminated from the neural plate, while premigratory NCC are not labeled. Thus, our Mef2c-F10N-LacZ and Mef2c-F10N-Cre transgenic mice provide new resources for tracing migratory NCC and analyzing gene function in migrating and differentiating NCC independently of NCC formation.

Expression of the bacterial beta-galactosidase reporter gene (lacZ) in the vector used for the Knockout Mouse Project (KOMP) is driven by the endogenous promoter of the target gene. In tissues from KOMP mice, histochemical staining for LacZ enzyme activity can be used to determine gene expression patterns. With this technique, we have produced a comprehensive resource of gene expression using both whole mount (WM) and frozen section (FS) LacZ staining in 313 unique KOMP mutant mouse lines. Of these, ∼ 80% of mutants showed specific staining in one or more tissues, while ∼ 20% showed no specific staining, ∼ 13% had staining in only one tissue, and ∼ 25% had staining in >6 tissues. The highest frequency of specific staining occurred in the brain (∼ 50%), male gonads (42%), and kidney (39%). The WM method was useful for rapidly identifying whole organ and some substructure staining, while the FS method often revealed substructure and cellular staining specificity. Both staining methods had >90% repeatability in biological replicates. Nonspecific LacZ staining occurs in some tissues due to the presence of bacteria or endogenous enzyme activity. However, this can be effectively distinguished from reporter gene activity by the combination of the WM and FS methods. After careful annotation, LacZ staining patterns in a high percentage of mutants revealed a unique structure-function not previously reported for many of these genes. The validation of methods for LacZ staining, annotation, and expression analysis reported here provides unique insights into the function of genes for which little is currently known.

Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown.

The lac operon is one of the best known gene regulatory circuits and constitutes a landmark example of how bacteria tune their metabolism to nutritional conditions. It is nearly ubiquitous in Escherichia coli strains justifying the use of its phenotype, the ability to consume lactose, for species identification. Lactose is the primary sugar found in milk, which is abundant in mammals during the first weeks of life. However, lactose is virtually non-existent after the weaning period, with humans being an exception as many consume dairy products throughout their lives. The absence of lactose during adulthood in most mammals and the rarity of lactose in the environment, means that the selective pressure for maintaining the lac operon could be weak for long periods of time. Despite the ability to metabolize lactose being a hallmark of E. coli’s success when colonizing its primary habitat, the mammalian intestine, the selective value of this trait remains unknown in this ecosystem during adulthood. Here we determine the competitive advantage conferred by the lac operon to a commensal strain of E. coli when colonizing the mouse gut. We find that its benefit, which can be as high as 11%, is contingent on the presence of lactose in the diet and on the presence of other microbiota members in the gut, but the operon is never deleterious. These results help explaining the pervasiveness of the lac operon in E. coli, but also its polymorphism, as lac-negative E. coli strains albeit rare can naturally occur in the gut.

The origin of mutations under selection has been intensively studied using the Cairns-Foster system, in which cells of an Escherichia coli lac mutant are plated on lactose and give rise to 100 Lac+ revertants over several days. These revertants have been attributed variously to stress-induced mutagenesis of nongrowing cells or to selective improvement of preexisting weakly Lac+ cells with no mutagenesis. Most revertant colonies (90%) contain stably Lac+ cells, while others (10%) contain cells with an unstable amplification of the leaky mutant lac allele. Evidence is presented that both stable and unstable Lac+ revertant colonies are initiated by preexisting cells with multiple copies of the F'lac plasmid, which carries the mutant lac allele. The tetracycline analog anhydrotetracycline (AnTc) inhibits growth of cells with multiple copies of the tetA gene. Populations with tetA on their F'lac plasmid include rare cells with an elevated plasmid copy number and multiple copies of both the tetA and lac genes. Pregrowth of such populations with AnTc reduces the number of cells with multiple F'lac copies and consequently the number of Lac+ colonies appearing under selection. Revertant yield is restored rapidly by a few generations of growth without AnTc. We suggest that preexisting cells with multiple F'lac copies divide very little under selection but have enough energy to replicate their F'lac plasmids repeatedly until reversion initiates a stable Lac+ colony. Preexisting cells whose high-copy plasmid includes an internal lac duplication grow under selection and produce an unstable Lac+ colony. In this model, all revertant colonies are initiated by preexisting cells and cannot be stress induced.

BackgroundPulmonary veno-occlusive disease is caused by excessive cell proliferation and fibrosis, which obliterate the lumen of pulmonary venules, leading to pulmonary hypertension, right ventricular failure, and death. This condition has no effective treatment and a 5-year survival of

Bis(2-ethylhexyl) phthalate, generally known as DEHP is a synthetic compound mainly used as a plasticizer to make polyvinyl chloride products flexible and soft. The present work aimed to study the toxicity of Bis(2-ethylhexyl) phthalate on the third instar larvae of transgenic Drosophila melanogaster(hsp70-lacZ) Bg 9 . The hsp70 gene is associated with the β-galactosidase in our present transgenic strain therefore, the more activity of β-galactosidase will indirectly correspond to hsp70 expression. The third instar larvae were allowed to feed on the diet for 24 h having 0.001, 0.005, 0.01, and 0.02 M of Bis(2-ethylhexyl) phthalate at the final concentration. After the exposure of 24hrs, the larvae were subjected to ONPG assay, X-gal staining, trypan blue exclusion test, oxidative stress markers assays, and comet assay. A dose-dependent increase in hsp70 expression, tissue damage, Glutathione-S-transferase (GST) activity, lipid peroxidation, monoamine oxidase, caspase-9 & 3, protein carbonyl content (PCC), DNA damage and decrease in the glutathione (GSH) content, delta-aminolevulinic acid dehydrogenase (ẟ-ALD-D) and acetylcholinesterase activity were observed in the larvae exposed to 0.005, 0.01, 0.02 M of Bis-(2-ethylhexyl) phthalate. The dose of 0.001 M of Bis(2-ethylhexyl) phthalate did not showed any toxic effects and hence can be considered as No Observed Adverse Effect Level (NOAEL) for Bis(2-ethylhexyl) phthalate. The study supports the use of Drosophila for the evaluation of possible toxic effects associated with synthetic compounds., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Kajal Gaur reports was provided by Aligarh Muslim University., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The occurrence of DNA looping is ubiquitous. This process plays a well-documented role in the regulation of prokaryotic gene expression, such as in regulation of the Escherichia coli lactose (lac) operon. Here we present two complementary methods for high-resolution in vivo detection of DNA/protein binding within the bacterial nucleoid by using either chromatin immunoprecipitation combined with phage λ exonuclease digestion (ChIP-exo) or chromatin endogenous cleavage (ChEC), coupled with ligation-mediated polymerase chain reaction (LM-PCR) and Southern blot analysis. As an example, we apply these in vivo protein-mapping methods to E. coli to show direct binding of architectural proteins in the Lac repressor-mediated DNA repression loop., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Genes encoding biosynthetic enzymes that make ergosterol, the major fungal membrane sterol, are regulated, in part, at the transcriptional level. Two transcription factors, Upc2p and Ecm22p, bind to the promoters of most ergosterol biosynthetic (ERG) genes, including ERG2 and ERG3, and activate these genes upon sterol depletion. We have identified the transcriptional activation domains of Upc2p and Ecm22p and found that UPC2-1, a mutation that allows cells to take up sterols aerobically, increased the potency of the activation domain. The equivalent mutation in ECM22 also greatly enhanced transcriptional activation. The C-terminal regions of Upc2p and Ecm22p, which contained activation domains, also conferred regulation in response to sterol levels. Hence, the activation and regulatory domains of these proteins overlapped. However, the two proteins differed markedly in how they respond to an increased need for sterols. Upon inducing conditions, Upc2p levels increased, and chromatin immunoprecipitation experiments revealed more Upc2p at promoters even when the activation/regulatory domains were tethered to a different DNA-binding domain. However, induction resulted in decreased Ecm22p levels and a corresponding decrease in the amount of Ecm22p bound to promoters. Thus, these two activators differ in their contributions to the regulation of their targets.