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Expression of recombinant genes can be controlled using inducible promoters. However, the most commonly used IPTG- and arabinose-inducible promoters result in an 'all-or-nothing' response, leading to fully induced and uninduced bacterial subpopulations. Here, we investigate whether appropriate modifications to these promoter systems can be combined into a single vector system, enabling homogenous expression of two genes of interest that can be precisely tuned using inducer concentration. We show that modifications of positive feedback loops related to inducer uptake result in homogeneous gene expression in both the T7 lactose and pBAD arabinose systems. Furthermore, these two modified systems were combined into a single vector, pRAT-sfGFP that provides the desired tunable expression of two genes of interest. Finally, we test this single-vector system as a tool for studying two-component genetic circuits, using toxin-antitoxin modules as model systems. This novel low-copy single vector expression system opens up new possibilities for investigating the function of two-component bacterial genetic circuits. [ABSTRACT FROM AUTHOR]

pET vectors allow inducible expression of recombinant proteins in Escherichia coli. In this system, isopropyl β- d -1-thiogalactopyranoside (IPTG) drives lac UV5 promoter to produce T7 RNA polymerase, simultaneously releasing the suppression of T7 lac 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. [ABSTRACT FROM AUTHOR]

The continuous emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants associated with the adaptive evolution of the virus is prolonging the global coronavirus disease 2019 (COVID-19) pandemic. The modification of neutralizing antibodies based on structural information is expected to be a useful approach to rapidly combat emerging variants. A dimerized variable domain of heavy chain of heavy chain antibody (VHH) P17 that has highly potent neutralizing activity against SARS-CoV-2 has been reported but the mode of interaction with the epitope remains unclear. Here, we report the X-ray crystal structure of the complex of monomerized P17 bound to the SARS-CoV-2 receptor binding domain (RBD) and investigated the binding activity of P17 toward various variants of concern (VOCs) using kinetics measurements. The structure revealed details of the binding interface and showed that P17 had an appropriate linker length to have an avidity effect and recognize a wide range of RBD orientations. Furthermore, we identified mutations in known VOCs that decrease the binding affinity of P17 and proposed methods for the acquisition of affinity toward the Omicron RBD because Omicron is currently the most predominant VOC. This study provides information for the rational design of effective VHHs for emerging VOCs. [ABSTRACT FROM AUTHOR]

AIMP2-DX2 is a splicing variant of AIMP2 protein which has been implicated in human lung cancer and chemoresistance of ovarian cancer (J.W. Choi, D.G. Kim, A.E. Lee, H.R. Kim, J.Y. Lee, N.H. Kwon, et al., 2011; J.W. Choi, J.W. Lee, J.K. Kim, H.K. Jeon, J.J. Choi, D.G. Kim, et al., 2012) [1,2]. We have shown, here, the data for the expression of AIMP2-DX2 protein in Escherichia coli and optimization of the critical steps in purification of AIMP2-DX2. The data described here has been successfully used to get a maximum yield of highly pure AIMP2-DX2 for subsequent characterization of its biophysical property in: "Purification and biophysical characterization of the AIMP2-DX2 protein" (R. Jha, H.Y. Cho, A. Ul Mushtaq, K. Lee, D.G. Kim, S. Kim, et al., 2017) [3].

Synthetic dsRNA are valuable tools for reverse genetics research and virus silencing applications. Its synthesis can be performed both in vivo or in vitro. Whilst the latter presents the drawback of high production cost, the former has the advantage of being less expensive and suitable for scalable production. In general, dsRNAs are obtained in vivo from Escherichia coli heterologous systems that require the gene for the T7 RNA polymerase inducible by IPTG. The (ds)RNAs for gene of interest are then synthesized under the T7 promoter. In this work, we present a reliable vector system that includes the insulated promoter proD for the constitutive expression of dsRNA in E. coli that does not require any inducer and that renders elevated dsRNA yield. In tandem, the T7 and proD promoters render the highest dsRNA yield. The accumulation of dsRNA in this system entails a high metabolic cost for the cell. Bacterial RNA extractions that included dsRNAs homologous to the m5GFPer gene and derived from both the synthetic and constitutive promoters induce silencing of GFP expression in Nicotiana benthamiana 16c. Key points • A vector system that includes a constitutive promoter and a T7 promoter in tandem for maximizing dsRNA synthesis. • The metabolic cost for bacteria is maximum when the two promoters are operating simultaneously and results from the accumulation of dsRNA. • Bacterial RNA extractions from both the induced and constitutive systems that include a mGFP5er-derived dsRNA are capable of silencing the GFP expression in Nicotiana benthamiana 16c plants [ABSTRACT FROM AUTHOR]

CRISPR/Cas systems act to protect the cell from invading nucleic acids in many bacteria and archaea. The bacterial immune protein Cas9 is a component of one of these CRISPR/Cas systems and has recently been adapted as a tool for genome editing. Cas9 is easily targeted to bind and cleave a DNA sequence via a complementary RNA; this straightforward programmability has gained Cas9 rapid acceptance in the field of genetic engineering. While this technology has developed quickly, a number of challenges regarding Cas9 specificity, efficiency, fusion protein function, and spatiotemporal control within the cell remain. In this work, we develop a platform for constructing novel proteins to address these open questions. We demonstrate methods to either screen or select active Cas9 mutants and use the screening technique to isolate functional Cas9 variants with a heterologous PDZ domain inserted within the protein. As a proof of concept, these methods lay the groundwork for the future construction of diverse Cas9 proteins. Straightforward and accessible techniques for genetic editing are helping to elucidate biology in new and exciting ways; a platform to engineer new functionalities into Cas9 will help forge the next generation of genome-modifying tools.

Abstract Background Escherichia coli is one of the most widely used hosts for recombinant protein production in academia and industry. Strain BL21(DE3) is frequently employed due to its advantageous feature of lacking proteases which avoids degradation of target protein. Usually it is used in combination with the T7-pET system where induction is performed by one point addition of IPTG. We recently published a few studies regarding lactose induction in BL21(DE3) strains. BL21(DE3) can only take up the glucose-part of the disaccharide when fed with lactose. However, initially additional glucose has to be supplied as otherwise the ATP-related lactose uptake barely happens. Yet, as lactose is an inexpensive compound compared to glucose and IPTG, a new induction strategy by a lactose-only feed during induction seems attractive. Thus, we investigated this idea in the galactose metabolizing strain HMS174(DE3). Results We show that strain HMS174(DE3) can be cultivated on lactose as sole carbon source during induction. We demonstrate that strain HMS174(DE3) exhibits higher product and biomass yields compared to BL21(DE3) when cultivated in a lactose fed-batch. More importantly, HMS174(DE3) cultivated on lactose even expresses more product than BL21(DE3) in a standard IPTG induced glucose fed-batch at the same growth rate. Finally, we demonstrate that productivity in HMS174(DE3) lactose-fed batch cultivations can easily be influenced by the specific lactose uptake rate (qs,lac). This is shown for two model proteins, one expressed in soluble form and one as inclusion body. Conclusions As strain HMS174(DE3) expresses even slightly higher amounts of target protein in a lactose fed-batch than BL21(DE3) in a standard cultivation, it seems a striking alternative for recombinant protein production. Especially for large scale production of industrial enzymes cheap substrates are essential. Besides cost factors, the strategy allows straight forward adjustment of specific product titers by variation of the lactose feed rate.

Abstract Background Transposon mutagenesis is highly valuable for bacterial genetic and genomic studies. The transposons are usually delivered into host cells through conjugation or electroporation of a suicide plasmid. However, many bacterial species cannot be efficiently conjugated or transformed for transposon saturation mutagenesis. For this reason, temperature-sensitive (ts) plasmids have also been developed for transposon mutagenesis, but prolonged incubation at high temperatures to induce ts plasmid loss can be harmful to the hosts and lead to enrichment of mutants with adaptive genetic changes. In addition, the ts phenotype of a plasmid is often strain- or species-specific, as it may become non-ts or suicidal in different bacterial species. Results We have engineered several conditional suicide plasmids that have a broad host range and whose loss is IPTG-controlled. One construct, which has the highest stability in the absence of IPTG induction, was then used as a curable vector to deliver hyperactive miniTn5 transposons for insertional mutagenesis. Our analyses show that these new tools can be used for efficient and regulatable transposon mutagenesis in Escherichia coli, Acinetobacter baylyi and Pseudomonas aeruginosa. In P. aeruginosa PAO1, we have used this method to generate a Tn5 insertion library with an estimated diversity of ~ 108, which is ~ 2 logs larger than the best transposon insertional library of PAO1 and related Pseudomonas strains previously reported. Conclusion We have developed a number of IPTG-controlled conditional suicide plasmids. By exploiting one of them for transposon delivery, a highly efficient and broadly useful mutagenesis system has been developed. As the assay condition is mild, we believe that our methodology will have broad applications in microbiology research.

A novel enzyme, thiourocanate hydratase, which catalyses the conversion of thiourocanic acid to 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid, was isolated from the ergothioneine-utilizing strain, Burkholderia sp. HME13. When the HME13 cells were cultured in medium containing ergothioneine as the sole nitrogen source, thiourocanate-metabolizing activity was detected in the crude extract from the cells. However, activity was not detected in the crude extract from HME13 cells that were cultured in Luria-Bertani medium. The gene encoding thiourocanate hydratase was cloned and expressed in Escherichia coli , and the recombinant enzyme was purified to homogeneity. The enzyme showed maximum activity at pH 7.5 and 55°C and was stable between pH 5.0 and 10.5, and at temperatures up to 45°C. The K m and V max values of thiourocanate hydratase towards thiourocanic acid were 30 μM and 7.1 μmol/min/mg, respectively. The enzyme was strongly inhibited by CuCl2 and HgCl2. The amino acid sequence of the enzyme showed 46% identity to urocanase from Pseudomonas putida , but thiourocanate hydratase had no urocanase activity. [ABSTRACT FROM AUTHOR]

Lactic acid bacteria (LAB) are important for many biotechnological applications such as bioproduction and engineered probiotics for therapy. Inducible promoters are key gene expression control elements, yet those available in LAB are mainly based on bacteriocin systems and have many drawbacks, including large gene clusters, costly inducer peptides, and little portability to in vivo settings. Using Lactobacillus gasseri , a model commensal bacteria from the human gut, we report the engineering of synthetic LactoSpanks promoters (Pls), a collection of variable strength inducible promoters controlled by the LacI repressor from E. coli and induced by isopropyl β-d-1-thiogalactopyranoside (IPTG). We first show that the Phyper-spank promoter from Bacillus subtilis is functional in L. gasseri , albeit with substantial leakage. We then construct and screen a semirational library of Phyper-spank variants to select a set of four IPTG-inducible promoters that span a range of expression levels and exhibit reduced leakages and operational dynamic ranges (from ca. 9 to 28 fold-change). With their low genetic footprint and simplicity of use, LactoSpanks will support many applications in L. gasseri , and potentially other lactic acid and Gram-positive bacteria.

Often glycosidase assays are based on small-molecule compounds where a glycan of interest is linked to a chromophore allowing for easy detection of cleavage of the glycoside bond. However, such compounds only resemble part of the more complex substrate molecule for enzymes acting on glycoconjugates of glycopeptides or glycoproteins. Nonetheless, the advantage is obvious as enzyme activity is readily recorded and kinetic parameters easily obtained. This is not often the case with glycopeptides or glycoproteins as these may reveal increased complexity in terms of heterogeneity in protein-glycan stoichiometry and restricted enzyme accessibility. However, a kinetic analysis of glycan release from glycopeptides could provide information complementary to that of small-molecule substrates, especially if providing kinetic parameters that are immediately comparable. We have characterized the steady state kinetics of wild type and mutant variants of Bifidobacterium longum endo-α- N -acetylgalactosaminidase, by recording the enzymatic release of Galβ(1–3)GalNAc from bovine glycomacropeptide pre-treated with sialidase to remove sialic acid units. Differences between previously reported kinetic constants obtained with synthetic substrates and those obtained in the present work demonstrate an influence of the peptide moiety on the kinetic properties of endo-α- N -acetylgalactosaminidase. The devised assay and data handling method determines the accessible substrate concentration as well as the steady state kinetic parameters, K M and k cat , for glycoconjugates of glycopeptides described by the same units as obtained from using small-molecule substrates and thus allows for a direct comparison. Image 1 • Assay for enzymatic O-linked 2- N -Acetyl hexosamine glycan release. • Analysis tools for quantitative data of enzymatic O-linked glycan release. • Preparation of glycopeptides for enzymes acting on O-linked hexosamine glycans. [ABSTRACT FROM AUTHOR]

Green fluorescent protein (GFP) is a globular protein used as biosensor and biomarker in medical and industrial fields. However, due to the expensive production costs of expressing proteins using high‐cost inducers like isopropyl‐β‐d‐1‐thiogalactopyranoside (IPTG), the number of GFP applications are still scarce. This work studied the production of enhanced GFP (EGFP) using Escherichia coli BL21 (DE3) [pLysS; pET28(a)], aiming to increase its yield and reduce costs. First, the influence of agitation rate, induction time, and concentration of IPTG in the production of EGFP was evaluated, but only the first two parameters were significant. Subsequently, aiming to reduce costs related to the use of inducer, the IPTG concentration (0.005, 0.010, and 0.025 mM) was decreased and, interestingly, the production levels were maintained or increased. These results show that a proper choice of production conditions, particularly through the decrease of inducer concentration, is effective to reduce the upstream production costs and guarantee high EGFP expression. [ABSTRACT FROM AUTHOR]

Since 1961, L-asparaginase has been used to treat patients with acute lymphocytic leukemia. It rapidly depletes the plasma asparagine and deprives the blood cells of this circulating amino acid, essential for the metabolic cycles of cells. In the search for viable alternatives to produce L-asparaginase, this work aimed to produce this enzyme from Escherichia coli in a shaker and in a 3 L bioreactor. Three culture media were tested: defined, semi-defined and complex medium. L-asparaginase activity was quantified using the β-hydroxamate aspartic acid method. The defined medium provided the highest L-asparaginase activity. In induction studies, two inducers, lactose and its analog IPTG, were compared. Lactose was chosen as an inducer for the experiments conducted in the bioreactor due to its natural source, lower cost and lower toxicity. Batch and fed-batch cultures were carried out to reach high cell density and then start the induction. Batch cultivation provided a final cell concentration of 11 g L−1 and fed-batch cultivation produced 69.90 g L−1 of cells, which produced a volumetric activity of 43,954.79 U L−1 after lactose induction. L-asparaginase was produced in a shaker and scaled up to a bioreactor, increasing 23-fold the cell concentration and thus, the enzyme productivity.

Our group has used the marine bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) as a platform for the successful recombinant production of “difficult” proteins, including eukaryotic proteins, at low temperatures. However, there is still room for improvement both in the refinement of PhTAC125 expression plasmids and in the bacterium’s intrinsic ability to accumulate and handle heterologous products. Here, we present an integrated approach of plasmid design and strain engineering finalized to increment the recombinant expression and optimize the inducer uptake in PhTAC125. To this aim, we developed the IPTG-inducible plasmid pP79 and an engineered PhTAC125 strain called KrPL LacY+. This mutant was designed to express the E. coli lactose permease and to produce only a truncated version of the endogenous Lon protease through an integration-deletion strategy. In the wild-type strain, pP79 assured a significantly better production of two reporters in comparison to the most recent expression vector employed in PhTAC125. Nevertheless, the use of KrPL LacY+ was crucial to achieving satisfying production levels using reasonable IPTG concentrations, even at 0 °C. Both the wild-type and the mutant recombinant strains are characterized by an average graded response upon IPTG induction and they will find different future applications depending on the desired levels of expression.

Jembrana diseases are caused by Jembrana Diseases Virus (JDV). The previous study showed that Jembrana Trans-Activator of Trancriptation (JTAT) recombinant protein is effective as a vaccine for Jembrana diseases. The production of JTAT protein needs to be optimized to obtain a higher amount of vaccine. High expression of JTAT protein will produce a high vaccine product. This study aimed to examine the effect of the addition of ethanol and IPTG in E. coli media on the expression of JTAT recombinant protein. This research was experimental research with factorial RAL design with a variation factor of ethanol and IPTG. Qualitatively, the induction of each IPTG, ethanol and interaction between the two could induce the expression of JTAT protein and could be identified with SDS-PAGE at ±11.8 kDa. Statistically, the induction of IPTG, ethanol and interaction between the two were not significantly different. Qualitative and quantitative data show that ethanol can induce JTAT protein expression. This result can be used as a preliminary study to test the effectiveness of ethanol as a substitute for IPTG in inducing the recombinant protein expression. [ABSTRACT FROM AUTHOR]

Leishmaniosis is a complex of diseases that can be fatal, if not given proper attention. Despite its relevance in the public health system, there is no vaccine capable of preventing the disease in humans so far and its treatment is expensive and aggressive to human health. The present study aims to optimize the induction parameters of the 503 Leishmania i. chagasi antigen expressed in recombinant Escherichia coli M15. The induction at different cell densities was evaluated in order to analyze the influence of the induction time on the yield of the protein of interest. In this segment, lactose and isopropyl-β-D-thiogalactopyranoside (IPTG) were used as inducer molecules, using various concentrations: 0.1 g/L, 1.0 g/L, and 10 g/L for lactose and 20 μM, 100 μM, 500 μM, and 1000 μM for IPTG. The results presented that the concentration of IPTG that obtained the higher antigen levels was that of 100 μM (0.087 g/L), a 10-fold lower concentration than was being previously used in this type of system and for lactose, it was 1 g/L (0.016 g/L). Thus, the induction with 100 μM allowed obtaining the antigen with a concentration 5.6 times higher than the lactose induction maximum concentration. [ABSTRACT FROM AUTHOR]

Background: Transposon mutagenesis is highly valuable for bacterial genetic and genomic studies. The transposons are usually delivered into host cells through conjugation or electroporation of a suicide plasmid. However, many bacterial species cannot be efficiently conjugated or transformed for transposon saturation mutagenesis. For this reason, temperature-sensitive (ts) plasmids have also been developed for transposon mutagenesis, but prolonged incubation at high temperatures to induce ts plasmid loss can be harmful to the hosts and lead to enrichment of mutants with adaptive genetic changes. In addition, the ts phenotype of a plasmid is often strain- or species-specific, as it may become non-ts or suicidal in different bacterial species. Results: We have engineered several conditional suicide plasmids that have a broad host range and whose loss is IPTG-controlled. One construct, which has the highest stability in the absence of IPTG induction, was then used as a curable vector to deliver hyperactive miniTn5 transposons for insertional mutagenesis. Our analyses show that these new tools can be used for efficient and regulatable transposon mutagenesis in Escherichia coli, Acinetobacter baylyi and Pseudomonas aeruginosa. In P. aeruginosa PAO1, we have used this method to generate a Tn5 insertion library with an estimated diversity of ~ 108, which is ~ 2 logs larger than the best transposon insertional library of PAO1 and related Pseudomonas strains previously reported. Conclusion: We have developed a number of IPTG-controlled conditional suicide plasmids. By exploiting one of them for transposon delivery, a highly efficient and broadly useful mutagenesis system has been developed. As the assay condition is mild, we believe that our methodology will have broad applications in microbiology research. [ABSTRACT FROM AUTHOR]

Background: Recombinant DNA technology enables us to produce proteins with desired properties and insubstantial amount for industrial applications. Endo-1, 4-β-glucanases (Egl) is one of the major enzyme involved in degradation of cellulose, an important component of plant cell wall. The present study was aimed at enhancing the production of endo-1, 4-β-glucanases (Egl) of Bacillus halodurans in Escherichia coli. Results: A putative Egl gene of Bacillus Halodurans was expressed in E. coli by cloning in pET 22b (+).On induction with isopropyl-b-D-1-thiogalactopyranoside, the enzyme expression reached upto ~20% of the cell protein producing 29.2 mg/liter culture. An increase in cell density to 12 in auto-inducing LB medium (absorbance at 600 nm) enhanced β-glucanase production up to 5.4 fold. The molecular mass of the enzyme was determined to be 39 KDa, which is nearly the same as the calculated value. Protein sequence was analyzed by CDD, Pfam, I TASSER, COACH, PROCHECK Servers and putative amino acids involved in the formation of catalytic, substrate and metal binding domains were identified. Phylogenetic analysis of the β-glucanases of B. halodurans was performed and position of Egl among other members of the genus Bacillus producing endo-glucanases was determined. Temperature and pH optima of the enzyme were found to be 60°C and 8.0, respectively, under the assay conditions. Conclusion: Production of endo-1, 4 β-glucanase enzymes fromB. halodurans increased several foldswhen cloned in pET vector and expressed in E. coli. To our knowledge, this is the first report of high-level expression and characterization of an endo-1, 4 β-glucanases from B. halodurans. [ABSTRACT FROM AUTHOR]

Recently, bacterial laccases has drawn researchers’ interest due to their ability to overcome high pH and salt concentration conditions compared to fungal laccases. Here we report a novel micro-aerobic cultivation strategy for enhancing CotA laccase expression and study its application for dye decolorization. Micro-aerobic cultivation of Escherichia coli BL21 (DE3) strain carrying pT7-FLAG-MAT-TAG-1-CotA had significantly enhanced CotA laccase activity up to 13903 U/L. The most unique findings of this investigation are that micro-aerobic cultivation strategy enhanced the reactive oxygen species production which consequently led to the over expression of CotA laccase gene. Malachite green, Crystal violet, Congo red and Bromophenol blue were efficiently decolorized by using purified CotA laccase without presence of any mediators at pH 6 and 9. These results provide a great platform for the dynamic production and application of bacterial laccase in industry. [ABSTRACT FROM AUTHOR]

Escherichia coli is commonly used for recombinant protein production with many available host strains. Screening experiments are often performed in batch mode using shake flasks and evaluating only the final product concentration. This conventional approach carries the risk of missing the best strain due to limited monitoring capabilities. Thus, this study focuses on investigating the general suitability of online respiration measurement for selecting expression hosts for heterologous protein production. The oxygen transfer rate (OTR) for different T7‐RNA polymerase‐dependent Escherichia coli expression strains was compared under inducing and noninducing conditions. As model enzymes, a lipase A from Bacillus subtilis (BSLA) and a 3‐hydroxybutyryl‐CoA dehydrogenase from Thermus thermophilus (HBD) were chosen. Four strains were compared during expression of both enzymes in autoinduction medium. Additionally, four strains were compared during expression of the BSLA with IPTG induction. It was found that the metabolic burden during recombinant protein production induces a phase of constant OTR, while undisturbed cell growth with no or little product formation is indicated by an exponential increase. This pattern is independent of the host strain, expressed enzyme, and induction method. Furthermore, the OTR gives information about carbon source consumption, biomass formation, and the transition from production to noninduced second growth phase, thereby ensuring a fair comparison of different strains. In conclusion, online monitoring of the respiration activity is suited to qualitatively identify, if a recombinant protein is produced by a strain or not. Furthermore, laborious offline sampling is avoided. Thus, the technique is easier and faster compared to conventional approaches. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:315–327, 2018 [ABSTRACT FROM AUTHOR]

Background: Biocatalytic production of L-phosphinothricin (L-PPT) is currently the most promising method. In this work, we use an Escherichia coli strain coexpressing of D-amino acid oxidase and catalase (E. coli DAAO-CAT) to oxidation biocatalytic D-PPT to PPO, then use the second E. coli strain coexpressing glutamate dehydrogenase and formate dehydrogenase (E. coli GluDH-FDH) to reduce biocatalytic PPO to L-PPT., Main Methods and Major Results: We compared the effects of different concentrations of IPTG or lactose on protein expression and enzyme activity in 5 L fermenter. The best induction conditions for E. coli DAAO-CAT were 0.05 mM IPTG, induction for 18 h at 28°C. The specific enzyme activities of DAAO and CAT were 153.20 U g -1 and 896.23 U g -1 , respectively. The optimal induction conditions for E. coli GluDH-FDH were 0.2 mM IPTG, induction for 19 h at 28°C. The specific enzyme activities of GluDH and FDH were 41.72 U g -1 and 109.70 U g -1 , respectively. The 200 mM D-PPT was biocatalyzed by E. coli DAAO-CAT for 4 h with space-time yield of 9.0 g·L -1 ·h -1 and conversion rate of over 99.0%. Then 220 mM PPO was converted to L-PPT by E. coli GluDH-FDH for 3 h with space-time yield of 14.5 g·L -1 ·h -1 and conversion rate of over 99.0%. To our knowledge, this is the most efficient biocatalytic reaction for L-PPT production., Conclusions and Implications: We found that IPTG has advantages compared with lactose in the enzyme activity and biomass of E. coli DAAO-CAT and E. coli GluDH-FDH, and IPTG is more environmentally friendly. Our data implicated that IPTG can replace lactose in terms of economic feasibility and effectiveness for scaled-up industrial fermentations., (© 2023 Wiley-VCH GmbH.)

Considering importance of N protein for study of viral pathogenesis or development of immunodiagnostic assay, wereported effects of several conditions on purity and homogeneity of recombinant SARS-CoV N protein expressed in E.coli BL21. The SARS-CoV N gene was reverse transcribed and amplified by the reverse transcription-polymerase chainreaction (RT-PCR) technique. The amplicons were cloned into pGEX-6P1 and followed by subcloning of the targetgene into pQE-80L. After inserting the recombinant plasmid (pQE80-N) into E. coli, the recombinant protein (6 x Histag-N protein fusion) was expressed by inducing the bacterial cells with 0.1-0.5 mM isopropyl-1-thio-Dgalactopyranoside(IPTG) for 1-5 h. The protein recombinant were extracted from the bacterial cells by NTT buffercontaining 0-20 mM imidazol, and followed by Ni-NTA affinity resin purification. The results showed that induction ofE. coli BL21 with 0.2 mM IPTG for 4 h and followed with lysis of bacterial cells in NTT buffer containing 10 mMimidazol were optimal conditions to obtain the pure recombinant SARS-CoV N protein.

A rapid antibody detection test is very useful for detection of lymphatic filariasis, especially for certification and surveillance of post-mass drug administration. One such kit, panLF RapidTM (commercialized by Malaysian BioDiagnostic Research Sdn. Bhd.) had been developed in our laboratory for the detection of all species of filarial infections. It is based on the detection of anti-filarial IgG4 antibodies that react with recombinant Brugia malayi antigens, BmR1 and BmSXP. In this study, the growth of recombinant bacteria that produce BmSXP was optimized under shake flask fermentation for high yield of the recombinant antigen. The optimizations involved selection of suitable growth medium, IPTG concentration and induction time. The medium that yielded the highest biomass as well as total protein was Terrific Broth (TB) medium, which is an undefined medium. Initiation of induction of protein expression was found to be best at mid-log phase (OD600 = 1.5), with IPTG concentration of 1.0 mM, and harvest time at 9 h post-induction. This study showed that under the optimized conditions, the shake flask culture produced 4 g/L biomass (dry cell weight) of recombinant Escherichia coli BmSXP/pPROEXHTa/TOP10F’, which yielded 2.42 mg/L of purified BmSXP recombinant antigen. The purified antigen was analyzed by SDS-PAGE and the antigenicity of protein was confirmed by Western blot.

Synthetic biological systems have been utilized to develop a wide range of genetic circuits and components that enhance the performance of biosensing systems. Among them, cell-free systems are emerging as important platforms for synthetic biology applications. Genetic circuits play an essential role in cell-free systems, mainly consisting of sensing modules, regulation modules, and signal output modules. Currently, fluorescent proteins and aptamers are commonly used as signal outputs. However, these signal output modes cannot simultaneously achieve faster signal output, more accurate and reliable performance, and signal amplification. Ribozyme is a highly structured and catalytic RNA molecule that can specifically recognize and cut specific substrate sequences. Here, by adopting ribozyme as the signal output, we developed a cell-free biosensing genetic circuit coupled with the ribozyme cleavage reaction, enabling rapid and sensitive detection of small molecules. More importantly, we have also successfully constructed a 3D-printed sensor array and thereby achieved high-throughput analysis of an inhibitory drug. Furthermore, our method will help expand the application range of ribozyme in the field of synthetic biology and also optimize the signal output system of cell-free biosensing, thus promoting the development of cell-free synthetic biology in biomedical research, clinical diagnosis, environmental monitoring, and food inspection.

Ralstonia eutropha H16 ( Cupriavidus necator H16) is a Gram-negative, facultative chemolithoautotrophic bacterium which can use H 2 and CO 2 as sole energy and carbon sources in the absence of organic substrates. The biotechnological use of R. eutropha H16 on an industrial scale has already been established; however, only a small number of tools promoting inducible gene expression is available. Within this study two systems promoting inducible expression were designed on the basis of the strong j5 promoter and the Escherichia coli lacI or the Pseudomonas putida cumate regulatory elements. Both expression vectors display desired regulatory features and further increase the number of suitable inducible expression systems for the production of metabolites and proteins with R. eutropha H16. [ABSTRACT FROM AUTHOR]

Recombinant consensus interferon (CIFN) is a therapeutic protein with molecular weight of 19.5 kDa having broad spectrum antiviral activity. Recombinant human CIFN (rhCIFN) has previously been expressed in Escherichia coli using isopropyl-β- d-thiogalactopyranoside (IPTG), a non-metabolizable and expensive compound, as inducer. For economical and commercial-scale recombinant protein production, it is greatly needed to increase the product yield in a limited time frame to reduce the processing cost. To reduce the cost of production of rhCIFN in E. coli, induction was accomplished by using lactose instead of IPTG. Lactose induction (14 g/L) in shake flask experiment resulted in higher yield as compared with 1 mM IPTG. Finally, with single-step purification on DEAE sepharose, 150 mg/L of >98% pure rhCIFN was achieved. In the present study, an attempt was made to develop a low cost process for producing quality product with high purity. Methods devised may be helpful for pilot-scale production of recombinant proteins at low cost. [ABSTRACT FROM AUTHOR]

Background: Engineering lactic acid bacteria (LAB) is of growing importance for food and feed industry as well as for in vivo vaccination or the production of recombinant proteins in food grade organisms. Often, expression of a transgene is only desired at a certain time point or period, e.g. to minimize the metabolic burden for the host cell or to control the expression time span. For this purpose, inducible expression systems are preferred, though cost and availability of the inducing agent must be feasible. We selected the plasmid free strain Lactobacillus plantarum 3NSH for testing and characterization of novel inducible promoters/repressor systems. Their feasibility in recombinant protein production was evaluated. Expression of the reporter protein mCherry was monitored with the BioLector® micro-fermentation system. Results: Reporter gene mCherry expression was compared under the control of different promoter/repressor systems: PlacA (an endogenous promoter/repressor system derived from L. plantarum 3NSH), PxylA (a promoter/repressor system derived from Bacillus megaterium DSMZ 319) and PlacSynth (synthetic promoter and codon-optimized repressor gene based on the Escherichia coli lac operon). We observed that PlacA was inducible solely by lactose, but not by non-metabolizable allolactose analoga. PxylA was inducible by xylose, yet showed basal expression under non-induced conditions. Growth on galactose (as compared to exponential growth phase on glucose) reduced basal mCherry expression at non-induced conditions. PlacSynth was inducible with TMG (methyl β-D-thiogalactopyranoside) and IPTG (isopropyl β-D-1-thiogalactopyranoside), but also showed basal expression without inducer. The promoter PlacSynth was used for establishment of a dual plasmid expression system, based on T7 RNA polymerase driven expression in L. plantarum. Comparative Western blot supported BioLector® micro-fermentation measurements. Conclusively, overall expression levels were moderate (compared to a constitutive promoter). Conclusions: We evaluated different inducible promoters, as well as an orthologous expression system, for controlled gene expression in L. plantarum. Furthermore, here we provide proof of concept for a T7 RNA polymerase based expression system for L. plantarum. Thereby we expanded the molecular toolbox for an industrial relevant and generally regarded as safe (GRAS) strain. [ABSTRACT FROM AUTHOR]

Coupling transcription of a cloned gene to the lac operon with induction by isopropylthio-β-galactoside (IPTG) has been a favoured approach for recombinant protein expression using Escherichia coli as a heterologous host for more than six decades. Despite a wealth of experimental data gleaned over this period, a quantitative relationship between extracellular IPTG concentration and consequent levels of recombinant protein expression remains surprisingly elusive across a broad spectrum of experimental conditions. This is because gene expression under lac operon regulation is tightly correlated with intracellular IPTG concentration due to allosteric regulation of the lac repressor protein (lacY). An in-silico mathematical model established that uptake of IPTG across the cytoplasmic membrane of E. coli by simple diffusion was negligible. Conversely, lacY mediated active transport was a rapid process, taking only some seconds for internal and external IPTG concentrations to equalize. Optimizing kcat and KM parameters by targeted mutation of the galactoside binding site in lacY could be a future strategy to improve the performance of recombinant protein expression. For example, if kcat were reduced whilst KM was increased, active transport of IPTG across the cytoplasmic membrane would be reduced, thereby lessening the metabolic burden on the cell and expediating accumulation of recombinant protein. The computational model described herein is made freely available and is amenable to optimize recombinant protein expression in other heterologous hosts., One-Sentence Summary: A computational model made freely available to optimize recombinant protein expression in Escherichia coli other heterologous hosts., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Prethrombin-2 recombinantproduction in E. coli still facesa bottle neck due to formation of inclusion bodies. The level of gene transcription can be regulated by using appropriate concentration of IPTG inducer. This study was aimed to determine the effect of isopropyl-β-D-thiogalactopyranoside (IPTG) concentration on prethrombin-2 expression. The prethrombin-2 expression was induced by various concentrations of IPTG at 12 o C hosted by E. coli ER2566. The result show that the suitable IPTG concentration for induction ofprethrombin-2 was 0.010 mM. It was indicated fromthe 63-kDa protein band obtained from the soluble fraction on SDS-PAGE. The higher concentrations of IPTG increased the amount of protein in the insoluble fraction due to the increased of expression rate. It concluded that the IPTG concentration affect the rate ofprethrombin-2 expression. [ABSTRACT FROM AUTHOR]

This paper employs the root locus method to conduct a detailed investigation of the parameter regions that ensure bistability in a well-studied gene regulatory network namely, lac operon of Escherichia coli (E. coli). In contrast to previous works, the parametric bistability conditions observed in this study constitute a complete set of necessary and sufficient conditions. These conditions were derived by applying the root locus method to the polynomial equilibrium equation of the lac operon model to determine the parameter values yielding the multiple real roots necessary for bistability. The lac operon model used was defined as an ordinary differential equation system in a state equation form with a rational right hand side, and it was compatible with the Hill and Michaelis-Menten approaches of enzyme kinetics used to describe biochemical reactions that govern lactose metabolism. The developed root locus method can be used to study the steady-state behavior of any type of convergent biological system model based on mass action kinetics. This method provides a solution to the problem of analyzing gene regulatory networks under parameter uncertainties because the root locus method considers the model parameters as variable, rather than fixed. The obtained bistability ranges for the lac operon model parameters have the potential to elucidate the appearance of bistability for E. coli cells in in vivo experiments, and they could also be used to design robust hysteretic switches in synthetic biology. [ABSTRACT FROM AUTHOR]

We here describe an IPTG-inducible system that reveals that the lac repressor alone can function as a potent transmodulator to regulate gene expression in the fission yeast, Schizosaccharomyces pombe. This expression system is a derivative of the Sz. pombe nmt promoter, which normally is strongly repressed by thiamine. With appropriate positioning of a lac operator site ( lacO) downstream of the TATA-box, we show that gene expression from a chimeric nmt::lacO promoter can be regulated by the lac repressor up to two orders of magnitude in response to IPTG. The chimeric nmt::lacO promoter is rapidly induced and when GFP is used as a reporter; almost full induction is achieved 40 min after the addition of IPTG. Like the wild-type nmt promoter, the chimeric nmt::lacO is repressed by thiamine. This allows expression in a short and defined window, e.g. the S-phase of a synchronized cell population, by first adding IPTG to turn on expression, followed by addition of thiamine to switch off expression. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Bacteria indicators have been widely developed but still need complicated techniques, instruments, or well-trained users. This is a proof-of-concept study to develop a straightforward and colorimetric bacterial indicator using bacteriophages (phages) as bio-receptors and simple biochemical reactions for high sensitivity. Among the various phages, phage T4 with a high specificity to Escherichia coli was used. Hydrophilic polyvinyl alcohol and trehalose were used as matrix polymers, and the optimal formulation ratio was examined to enhance the phage stability in the indicator matrix. Its sensitivity and detection rate were improved by investigating the interactions between phage T4 and isopropyl β - d -thiogalactopyranoside. The final indicator showed high sensitivity detecting up to 10 CFU/mL after 11 h, even in the food application test such as drinking water and milk samples containing different fat contents. Although some challenges remain to satisfy the regulatory requirements for bacteria in foods and be applied to various foods, the introduced simple approach can pave the way to develop indicators to detect E. coli present in real food using phages. • E. coli indicator was developed using phage T4 as a bacteria-sensing probe. • Polyvinyl alcohol forms 3D porous matrix for phage incorporation into the indicator. • Trehalose showed a strong protective effect on phage under freeze-drying process. • Optimizing phage T4 and IPTG concentration improved the bacterial sensing ability. • E. coli indicator detected up to 10 CFU/ml E. coli cells in liquid-based foods. [ABSTRACT FROM AUTHOR]

Neurotensin receptor type 1 (NTS1) is a G protein‐coupled receptor (GPCR) that affects cellular responses by initiating a cascade of interactions through G proteins. The kinetic details for these interactions are not well‐known. Here, NTS1‐nanodisc‐Gαs and Gαi1 interactions were studied. The binding affinities of Gαi1 and Gαs to NTS1 were directly measured by surface plasmon resonance (SPR) and determined to be 15 ± 6 nM and 31 ± 18 nM, respectively. This SPR configuration permits the kinetics of early events in signalling pathways to be explored and can be used to initiate descriptions of the GPCR interactome.Little is known of the kinetics of interactions between GPCRs and their signalling partners. NTS1 binds Gαi1 and Gαs with affinities of 15±6nM and 31±18nM (SE), respectively. This SPR assay may be applicable to multiple partners in the signalling cascade. We provide the first direct evidence for GPCR‐G protein coupling in nanodiscs. [ABSTRACT FROM AUTHOR]

An unsteady, unstructured, unsegregated and based on first principles mathematical model has been proposed to describe IPTG (isopropyl-β- d -tiogalactopiranoside) transport in induced fed-batch cultures of E. coli M15 Δ glyA [pQEαβrham] [pREP4] producing rhamnulose 1-phosphate aldolase (RhuA). The model predicts extracellular and intracellular IPTG concentration. Experimental extracellular IPTG concentrations under different operational conditions were obtained by HPLC–MS analysis. These experimental data were used to fit the parameters of the model. The model was also able to predict the experimental behavior of two different E. coli strains producing fuculose 1-phosphate aldolase (FucA). IPTG transport to cells was the contribution of three processes: a diffusion process, and two active processes (one non-specific and another specific). [ABSTRACT FROM AUTHOR]

Homo sapiens ECT2 is a cell cycle regulator that plays critical roles in cytokinesis. ECT2 activity is restrained during interphase via intra‐molecular interactions that involve its N‐terminal triple‐BRCT‐domain and its C‐terminal DH–PH domain. At anaphase, this self‐inhibitory mechanism is relieved by Plk1‐phosphorylated CYK‐4, which directly engages the ECT2 BRCT domain. To provide a structural perspective for this auto‐inhibitory property, we solved the crystal structure of the ECT2 triple‐BRCT‐domain. In addition, we systematically analyzed the interaction between the ECT2 BRCT domains with phospho‐peptides derived from its binding partner CYK‐4, and have identified Ser164 as the major phospho‐residue that links CYK‐4 to the second ECT2 BRCT domain.We solved the crystal structure of triple‐BRCT‐domain of ECT2. We show that phosphorylation of Ser164 in CYK‐4 is crucial for the interaction with ECT2. A canonical binding pocket in the second BRCT domain of ECT2 is proved to bind to pS164 in CYK‐4. The interactions between ECT2 and CYK‐4 can relieve the auto‐inhibition of ECT2. [ABSTRACT FROM AUTHOR]

Bacteria have adapted their NhaA Na+/H+ exchangers responsible for salt homeostasis to their different habitats. We present an electrophysiological and kinetic analysis of NhaA from Helicobacter pylori and compare it to the previously investigated exchangers from Escherichia coli and Salmonella typhimurium. Properties of all three transporters are described by a simple model using a single binding site for H+ and Na+. We show that H. pylori NhaA only has a small acidic shift of its pH‐dependent activity profile compared to the other transporters and discuss why a more drastic change in its pH activity profile is not physiologically required.The H. pylori NhaA Na+/H+ exchanger was previously reported to be pH‐insensitive. We investigate H. pylori NhaA by SSM‐based electrophysiology. H. pylori NhaA shows a similar profile to E. coli NhaA with a small acidic pK shift. NhaA exchangers from enterobacteria can be explained by the same mechanism. [ABSTRACT FROM AUTHOR]

Highlights: [•] Analysis of outer surface proteins of Bacillus subtilis as carrier proteins. [•] Recombinant proteins will not have to cross any membrane. [•] Fusion proteins can be expressed from an inducible promoter. [ABSTRACT FROM AUTHOR]

Background Inducible promoters are widely spread genetic tools for triggering, tuning and optimizing the expression of recombinant genes in engineered biological systems. Most of them are controlled by the addition of a specific exogenous chemical inducer that indirectly regulates the promoter transcription rate in a concentration-dependent fashion. In order to have a robust and predictable degree of control on promoter activity, the degradation rate of such chemicals should be considered in many applications like recombinant protein production. Results In this work, we use whole-cell biosensors to assess the half-life of three commonly used chemical inducers for recombinant Escherichia coli: Isopropyl β-D-1-thiogalactopyranoside (IPTG), anhydrotetracycline (ATc) and N-(3-oxohexanoyl)-L-homoserine lactone (HSL). A factorial study was conducted to investigate the conditions that significantly contribute to the decay rate of these inducers. Temperature has been found to be the major factor affecting ATc, while medium and pH have been found to highly affect HSL. Finally, no significant degradation was observed for IPTG among the tested conditions. Conclusions We have quantified the decay rate of IPTG, ATc and HSL in many conditions, some of which were not previously tested in the literature, and the main effects affecting their degradation were identified via a statistics-based framework. Whole-cell biosensors were successfully used to conduct this study, yielding reproducible measurements via simple multiwell-compatible assays. The knowledge of inducer degradation rate in several contexts has to be considered in the rational design of synthetic biological systems for improving the predictability of induction effects, especially for prolonged experiments. [ABSTRACT FROM AUTHOR]

Background: Poly3-hydroxyalkanoates (PHAs) are potential candidates for the industrial production of biodegradable plastics. Therefore, in the present study, expression and activity of one of the enzymes involved in the PHA synthesis, phaC2 (isolated from Pseudomonas aeruginosa PTCC1310), were investigated in Fad B deleted Escherichia coli. Materials and Methods: The inserts obtained from recombinant pTZ57R plasmids were ligated into the pGEX-5x-1 expression vector and then transformed into Fad B deleted E. coli cells using the heat shock method. This protein was then expressed using isopropyl beta-d-thiogalactoside (IPTG) as an inducer. By changing expression conditions such as IPTG and glucose concentration, time and temperature of incubation with IPTG, the expression conditions were optimized. Results: The optimum condition for the expression of this enzyme was: 1.5 mM IPTG, 1 mM glucose, incubated at 37°C for 2 hours. Conclusion: We obtained functional expression of the phaC2 gene and investigated various conditions that could influence the expression of protein to optimize production of PHA synthase enzymes. This would allow us to study PHA production in large quantities.

Studies have shown that the production of unnecessary proteins burdens the cellular growth mainly due to allocation of cellular resources to unnecessary protein synthesis, thereby limiting the resources available for growth. In the current study, we focus on the effect of pre-induction and nutritional status of the medium on the burden imposed on growth due to the synthesis of unnecessary protein. Escherichia coli cells with different history were grown in a glycerol media with and without IPTG to characterize the burden imposed due to the synthesis of β-galactosidase. Effect of pre-induced lac operon on growth and β-galactosidase expression on lactose milieu was also investigated. The study demonstrates that pre-induction has a strong influence on the extent of burden and is sustained in several generations. Further, the burden was much lower in a rich media relative to that observed in a minimal media. [ABSTRACT FROM AUTHOR]