lacZ Research Articles

X-ray crystal structure of proliferating cell nuclear antigen 1 from Aeropyrum pernix.

Proliferating cell nuclear antigen (PCNA) plays a critical role in DNA replication by enhancing the activity of various proteins involved in replication. Inthis study, the crystal structure of ApePCNA1, one of three PCNAs from thethermophilic archaeon Aeropyrum pernix, was elucidated. ApePCNA1 was cloned and expressed in Escherichia coli and the protein was purified and crystallized. The resulting crystal structure determined at 2.00 Å resolution revealed that ApePCNA1 does not form a trimeric ring, unlike PCNAs from other domains of life. It has unique structural features, including a long interdomain-connecting loop and a PIP-box-like sequence at the N-terminus, indicating potential interactions with other proteins. These findings provide insights into the functional mechanisms of PCNAs in archaea and their evolutionary conservation across different domains of life. A modified medium and protocol were used to express recombinant protein containing the lac operon. The expression of the target protein increased and the total incubation time decreased when using this system compared with those of previous expression protocols.

Identification of Grape Laccase Genes and Their Potential Role in Secondary Metabolite Synthesis.

Laccase, a copper-containing oxidoreductase, has close links with secondary metabolite biosynthesis in plants. Its activity can affect the synthesis and accumulation of secondary metabolites, thereby influencing plant growth, development, and stress resistance. This study aims to identify the grape laccases (VviLAC) gene family members in grape (Vitis vinifera L.) and explore the transcriptional regulatory network in berry development. Here, 115 VviLACs were identified and divided into seven (Type I-VII) classes. These were distributed on 17 chromosomes and out of 47 VviLACs on chromosome 18, 34 (72.34%) were involved in tandem duplication events. VviLAC1, VviLAC2, VviLAC3, and VviLAC62 were highly expressed before fruit color development, while VviLAC4, VviLAC12, VviLAC16, VviLAC18, VviLAC20, VviLAC53, VviLAC60 and VviLAC105 were highly expressed after fruit color transformation. Notably, VviLAC105 showed a significant positive correlation with important metabolites including resveratrol, resveratrol dimer, and peonidin-3-glucoside. Analysis of the transcriptional regulatory network predicted that the 12 different transcription factors target VviLACs genes. Specifically, WRKY and ERF were identified as potential transcriptional regulatory factors for VviLAC105, while Dof and MYB were identified as potential transcriptional regulatory factors for VviLAC51. This study identifies and provides basic information on the grape LAC gene family members and, in combination with transcriptome and metabolome data, predicts the upstream transcriptional regulatory network of VviLACs.

Extraordinary relative cooling power in the magnetocaloric La0.6Y0.07Ca0.33MnO3-δ

The magnetocaloric effect of La0.6Y0.07Ca0.33MnO3-δ is reported to cover a wide temperature range, including ambient temperature and cryogenic temperature, making it a promising material for magnetic refrigerators. Furthermore, the remarkable cooling power of La0.6Y0.07Ca0.33MnO3-δ is much higher than that of well-known magnetic refrigerants like Gd, (La–Sr)MnO3, (La–Sr)MnO3, (La–Ca–Pb)MnO3, and La1-xCaxMnO3 samples. La0.6Y0.07Ca0.33MnO3-δ can be used as a single magnetic refrigerant in MR at room-temperature and cryogenic temperatures. Consequently, La0.6Y0.07Ca0.33MnO3-δ is recommended as a potential magnetic refrigerant for various applications, including food refrigeration and nitrogen liquefaction.

Synthesis of di-rhamnolipids by the avirulent, mono-rhamnolipid producing strain Pseudomonas aeruginosa ATCC 9027.

To construct a derivative of the avirulent Pseudomonas aeruginosa ATCC 9027 that produces high levels of di-rhamnolipid, that has better physico-chemical characteristics for biotechnological applications than mono-rhamnolipid, which is the sole type produced by ATCC 9027. We used plasmids expressing the rhlC gene, which encodes for rhamnosyl transferase II that transforms mono- to di-rhamnolipids under different promoters and in combination with the gene coding for the RhlR quorum sensing regulator, or the mono-rhamnolipid biosynthetic rhlAB operon. The plasmids tested carrying the rhlC gene under the lac promoter were plasmid prhlC and prhlRC, while prhlAB-R-C expressed this gene from the rhlA promoter, forming part of the artificially constructed rhlAB-R-C operon. We measured rhamnolipds concentrations using the orcinol method and determined the proportion of mono-rhamnolipids and di-rhamnolipids by UPLC/MS/MS. We found that the expression of rhlC in P. aeruginosa ATCC 9027 caused the production of di-rhamnolipids and that the derivative carrying plasmid prhlAB-R-C gives the best results considering total rhamnolipids and a higher proportion of di-rhamnolipids. A P. aeruginosa ATCC 9027 derivative with increased di-rhamnolipids production was developed by expressing plasmid prhlAB-R-C, that produces similar rhamnolipids levels as PAO1 type-strain and presented a higher proportion of di-rhamnolipids than this type-strain.

Design of Thermoresponsive Genetic Controls with Minimal Heat-Shock Response.

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.

Engineered transcription activator-like effector dimer proteins confer DNA loop-dependent gene repression comparable to Lac repressor.

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.

Efficient heterologous expression of cellobiose 2-epimerase gene in Escherichia coli under the control of T7 lac promoter without addition of IPTG and lactose

In this study, the cellobiose 2-epimerase gene csce from Caldicellulosiruptor saccharolyticus was expressed in Escherichia coli using TB medium containing yeast extract Oxoid and tryptone Oxoid. Interesting, it was found that when the concentration of isopropyl-beta-d-thiogalactopyranoside (IPTG) and lactose was 0 (no addition), the activity of cellobiose 2-epimerase reached 5.88 U/mL. It was 3.70-fold higher than the activity observed when 1.0 mM IPTG was added. When using M9 medium without yeast extract Oxoid and tryptone Oxoid, cellobiose 2-epimerase gene could not be expressed without IPTG and lactose. However, cellobiose 2-epimerase gene could be expressed when yeast extract Oxoid or tryptone Oxoid was added, indicating that these supplements contained inducers for gene expression. In the absence of IPTG and lactose, the addition of soy peptone Angel-1 or yeast extract Angel-1 to M9 medium significantly upregulated the expression of cellobiose 2-epimerase gene in E. coli BL21 pET28a-csce, and these inductions led to higher expression levels compared to tryptone Oxoid or yeast extract Oxoid. The relative transcription level of csce was consistent with its expression level in E. coli BL21 pET28a-csce. In the medium TB without IPTG and lactose and containing yeast extract Angel-1 and soy peptone Angel-1, the activity of cellobiose 2-epimerase reached 6.88 U/mL, representing a 2.2-fold increase compared to previously reported maximum activity in E. coli. The significance of this study lies in its implications for efficient heterologous expression of recombinant enzyme proteins in E. coli without the need for IPTG and lactose addition.

Biosynthesis of Indigo Dyes and Their Application in Green Chemical and Visual Biosensing for Heavy Metals.

Fermentative production of natural colorants using microbial strains has emerged as a cost-effective and sustainable alternative to chemical synthesis. Visual pigments are used as signal outputs in colorimetric bacterial biosensors, a promising method for monitoring environmental pollutants. In this study, we engineered four self-sufficient indigo-forming enzymes, including HbpAv, bFMO, cFMO, and rFPMO, in a model bacterium E. coli. TrxA-bFMO was chosen for its strong ability to produce indigo under T7 lac and mer promoters' regulation. The choice of bacterial hosts, the supplementation of substrate l-tryptophan, and ventilation were crucial factors affecting indigo production. The indigo reporter validated the biosensors for Hg(II), Pb(II), As(III), and Cd(II). The biosensors reported Hg(II) as low as 14.1 nM, Pb(II) as low as 1.5 nM, and As(III) as low as 4.5 nM but increased to 25 μM for Cd(II). The detection ranges for Hg(II), Pb(II), As(III), and Cd(II) were quantified from 14.1 to 225 nM, 1.5 to 24.4 nM, 4.5 to 73.2 nM, and 25 to 200 μM, respectively. The sensitivity, responsive concentration range, and selectivity are comparable to β-galactosidase and luciferase reporter enzymes. This study suggests that engineered enzymes for indigo production have great potential for green chemical synthesis. Additionally, heterologous biosynthesis of indigo production can lead to the development of novel, low-cost, and mini-equipment bacterial biosensors with zero background noise for visual monitoring of pollutant heavy metals.

Multilevel regulation of lactose operon in Bacillus licheniformis: Coordination of LacR, CcpA and TnrA regulators

Previous research has confirmed the involvement of the lac1 gene cluster in lactose metabolism in Bacillus licheniformis, which is able to co-utilize lactose and low concentrations of glucose or certain carbon sources. However, the internal regulatory mechanisms underlying specific lactose utilization remain elusive. In this study, we conducted a comprehensive investigation to elucidate the modulation mechanism of the lac1 gene cluster. We discovered a 162 bp bidirectional promoter located between the lacDCAB and lacR genes, which encodes a GntR family transcriptional regulatory factor. A 38 bp perfectly palindromic sequence (BOX1) within this promoter is critical for inducing lac1 expression. LacR and CcpA were shown to bind specifically to BOX1, and their novel binding motifs were identified. In BL2ΔlacR, BL2ΔccpA, and BL2ΔlacRΔccpA mutant strains, we confirmed the differential inhibitory levels of LacR and CcpA on bidirectional promoters. Furthermore, it was demonstrated that TnrA also binds BOX1 and exerts a positive regulatory effect on the lac1 cluster. Finally, we proposed a novel model to unravel the complex molecular interactions controlling the expression of the lac1 gene. These insights provide a foundation for exploring rational design strategies to optimize lactose fermentation processes and improve the efficiency of industrial lactose utilization.

Multifaceted Roles of Ag+ Ions within and outside La–Ca–MnO3 Perovskite Manganites: Unveiling the Room Temperature Magnetocaloric Effect

Multifaceted Roles of Ag⁺ Ions within and outside La–Ca–MnO₃ Perovskite Manganites: Unveiling the Room Temperature Magnetocaloric Effect

Genome-wide analysis of the common bean (Phaseolus vulgaris) laccase gene family and its functions in response to abiotic stress

BackgroundLaccase (LAC) gene family plays a pivotal role in plant lignin biosynthesis and adaptation to various stresses. Limited research has been conducted on laccase genes in common beans.Results29 LAC gene family members were identified within the common bean genome, distributed unevenly in 9 chromosomes. These members were divided into 6 distinct subclades by phylogenetic analysis. Further phylogenetic analyses and synteny analyses indicated that considerable gene duplication and loss presented throughout the evolution of the laccase gene family. Purified selection was shown to be the major evolutionary force through Ka / Ks. Transcriptional changes of PvLAC genes under low temperature and salt stress were observed, emphasizing the regulatory function of these genes in such conditions. Regulation by abscisic acid and gibberellins appears to be the case for PvLAC3, PvLAC4, PvLAC7, PvLAC13, PvLAC14, PvLAC18, PvLAC23, and PvLAC26, as indicated by hormone induction experiments. Additionally, the regulation of PvLAC3, PvLAC4, PvLAC7, and PvLAC14 in response to nicosulfuron and low-temperature stress were identified by virus-induced gene silence, which demonstrated inhibition on growth and development in common beans.ConclusionsThe research provides valuable genetic resources for improving the resistance of common beans to abiotic stresses and enhance the understanding of the functional roles of the LAC gene family.

Advances in plastic mycoremediation: Focus on the isoenzymes of the lignin degradation complex

This study investigates P. ostreatus and A. bisporus biodegradation capacity of low density polyethylene (LDPE) oxidised to simulate environmental weathering. Fourier transform infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM) were used to analyse the degradation of LDPE treated with fungal cultures. Molecular implications of LDPE degradation by P. ostreatus and A. bisporus were evaluated by Reverse transcription followed by quantitative PCR (qRT-PCR) of lac, mnp and lip genes expression. After 90 days of incubation, FT-IR analysis showed, for both fungal treatments, an increasing in the intensity of peaks related to the asymmetric C-C-O stretching (1160 to 1000 cm−1) and the -OH stretching (3700 to 3200 cm−1) due to the formation of alcohols and carboxylic acid, indicating depolymerisation of LDPE. This was confirmed by the SEM analysis, where a widespread alteration of the surface morphology was observed for treated LDPE fragments. Results revealed that the exposure of P. ostreatus to oxidised LDPE treatment led to a significant increase in the expression of the lac6, lac7, lac9, lac10 and mnp2 genes, while A. bisporus showed an over-expression in lac2 and lac12 genes. The obtained results offer new perspectives for a biotechnological use of P. ostreatus and A. bisporus for plastic bioremediation.

The unreasonable effectiveness of equilibrium gene regulation through the cell cycle

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.

OptoLacI: optogenetically engineered lactose operon repressor LacI responsive to light instead of IPTG.

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.

3D printing of thick film NTC thermistor from preceramic polymer composites for ultra-high temperature measurement

Integrating thick/thin film sensors into component systems has emerged as a prevalent approach for monitoring in extreme environments. However, traditional vapor deposition methods face obstacles, including complex fabrication processes and the degradation of sensitive materials at extremely high temperatures. This work delineates the development of a polysilazane composite dual-layer thick-film Negative Temperature Coefficient (NTC) thermistor characterized by its suitability for extreme temperatures and robust bond strength achieved through an advanced near-net-shape printing methodology. High-temperature resistant La(Ca)CrO3/polysilazane films were printed as the sensitive layer, while a dense layer formed by Cr2O3/polysilazane was used as the protective layer. The bilayer structure resulted in a 2.5-fold increase in adhesion strength compared to the single-layer La(Ca)CrO3/polysilazane films. Experimental results indicate that the dual-layer thick-film NTC thermistor can be operated long-term at 1300 °C with a resistance drift rate of 0.9 %/h and survive short-term exposure to temperatures up to 1550 °C. As a proof of concept, this work applied 3D printing technology to fabricate a polysilazane composite dual-layer thick-film NTC thermistor on the surface of turbine blades and demonstrated its functionality under flame impingement at nearly 1300 °C. Such flexible 3D printing techniques pave the way for a new paradigm in manufacturing sensors capable of withstanding ultra-high temperatures.

Using origami and Shrinky Dinks to create active learning activities to tackle two microbiology concepts: cell structure differences and operon regulation.

This paper presents two low-cost hands-on activities designed to enhance student understanding and address the pedagogical challenges faced by microbiology professors in teaching concepts related to cell structure and gene regulation. In the first activity, we used Shrinky Dinks and Jeopardy-style game questions to explore the differences between prokaryotic and eukaryotic cells. Students have to collect pieces and physically build their cell models. The second activity uses origami organelles sets from Edvotek to illustrate the regulation of gene expression in the lac and trp operons, incorporating mutation scenarios for analysis. The intended audience comprises undergraduate students in microbiology, including biology, pre-medical studies, and health profession majors. The activities were deployed in three microbiology lectures, and students were surveyed. Students' feedback highlights the efficacy of the hands-on approach and increased class participation, as two of the recurring words in the students' survey were "helpful" and "fun."

Necessary and Sufficient Conditions for Event-Triggered Set Stabilizability of Markovian Jump Logical Networks.

This technical paper utilizes the Lyapunov theory to characterize the event-triggered set stabilizability of Markovian jump logical control networks (MJLCNs). Whereas the existing result for checking the set stabilizability of MJLCNs is only sufficient, this technical paper further establishes its necessary and sufficient condition. First, the Lyapunov function is established to describe the set stabilizability of MJLCNs necessarily and sufficiently by combining recurrent switching modes and desired state set. Then, the triggering condition and the input updating mechanism are designed regarding the value change of the Lyapunov function. Finally, the effectiveness of theoretical results is demonstrated by a biological example concerning the lac operon in Escherichia coli.

Evolutionary origin and gradual accumulation with plant evolution of the LACS family

BackgroundLACS (long-chain acyl-CoA synthetase) genes are widespread in organisms and have multiple functions in plants, especially in lipid metabolism. However, the origin and evolutionary dynamics of the LACS gene family remain largely unknown.ResultsHere, we identified 1785 LACS genes in the genomes of 166 diverse plant species and identified the clades (I, II, III, IV, V, VI) of six clades for the LACS gene family of green plants through phylogenetic analysis. Based on the evolutionary history of plant lineages, we found differences in the origins of different clades, with Clade IV originating from chlorophytes and representing the origin of LACS genes in green plants. The structural characteristics of different clades indicate that clade IV is relatively independent, while the relationships between clades (I, II, III) and clades (V, VI) are closer. Dispersed duplication (DSD) and transposed duplication (TRD) are the main forces driving the evolution of plant LACS genes. Network clustering analysis further grouped all LACS genes into six main clusters, with genes within each cluster showing significant co-linearity. Ka/Ks results suggest that LACS family genes underwent purifying selection during evolution. We analyzed the phylogenetic relationships and characteristics of six clades of the LACS gene family to explain the origin, evolutionary history, and phylogenetic relationships of different clades and proposed a hypothetical evolutionary model for the LACS family of genes in plants.ConclusionsOur research provides genome-wide insights into the evolutionary history of the LACS gene family in green plants. These insights lay an important foundation for comprehensive functional characterization in future research.

Hybrid stabilization of nonlinear systems based on a fully actuated system approach

This article studies the stabilization problem for a category of nonlinear systems. It introduces a novel hybrid control strategy specifically for nonlinear high-order fully actuated (HOFA) systems. The stabilization problem for these nonlinear HOFA systems is transformed into a stabilization problem for impulsive switched systems. This issue is addressed by applying impulsive control in discrete-time and switching control based on the HOFA system approach in continuous-time. Using switched Lyapunov functions, we obtain the criteria for the exponential and asymptotic stabilization of these systems. The effectiveness of this newly presented hybrid control strategy is exemplified by simulations of a robotic manipulator system and a reduced model of the lac operon.

Efficient overexpression and purification of severe acute respiratory syndrome coronavirus 2 nucleocapsid proteins in Escherichia coli.

The fundamental biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (Ncap), its use in diagnostic assays and its potential application as a vaccine component have received considerable attention since the outbreak of the Covid19 pandemic in late 2019. Here we report the scalable expression and purification of soluble, immunologically active, SARS-CoV-2 Ncap in Escherichia coli. Codon-optimised synthetic genes encoding the original Ncap sequence and four common variants with an N-terminal 6His affinity tag (sequence MHHHHHHG) were cloned into an inducible expression vector carrying a regulated bacteriophage T5 synthetic promoter controlled by lac operator binding sites. The constructs were used to express Ncap proteins and protocols developed which allow efficient production of purified Ncap with yields of over 200 mg per litre of culture media. These proteins were deployed in ELISA assays to allow comparison of their responses to human sera. Our results suggest that there was no detectable difference between the 6His-tagged and untagged original Ncap proteins but there may be a slight loss of sensitivity of sera to other Ncap isolates.