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.

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.

Galectins are a large and diverse protein family defined by the presence of a carbohydrate recognition domain (CRD) that binds β-galactosides. They play important roles in early development, tissue regeneration, immune homeostasis, pathogen recognition, and cancer. In many cases, studies that examine galectin biology and the effect of manipulating galectins are aided by, or require the ability to express and purify, specific members of the galectin family. In many cases, E. coli is employed as a heterologous expression system, and galectin expression is induced with isopropyl β-galactoside (IPTG). Here, we show that galectin-3 recognizes IPTG with micromolar affinity and that as IPTG induces expression, newly synthesized galectin can bind and sequester cytosolic IPTG, potentially repressing further expression. To circumvent this putative inhibitory feedback loop, we utilized an autoinduction protocol that lacks IPTG, leading to significantly increased yields of galectin-3. Much of this work was done within the context of a course-based undergraduate research experience, indicating the ease and reproducibility of the resulting expression and purification protocols.

Autoinduction systems in Escherichia coli can control the production of proteins without the addition of a particular inducer. In the present study, we optimized the heterologous expression of Moloney Murine Leukemia Virus derived Reverse Transcriptase (MMLV-RT) in E. coli. Among 4 autoinduction media, media Imperial College resulted the highest MMLV-RT overexpression in E. coli BL21 Star (DE3) with incubation time 96 h. The enzyme was produced most optimum in soluble fraction of lysate cells. The MMLV-RT was then purified using the Immobilized Metal Affinity Chromatography method and had specific activity of 629.4 U/mg. The system resulted lower specific activity and longer incubation of the enzyme than a classical Isopropyl ß-D-1-thiogalactopyranoside (IPTG)-induction system. However, the autoinduction resulted higher yield of the enzyme than the conventional induction (27.8%). Techno Economic Analysis revealed that this method could produce MMLV-RT using autoinduction at half the cost of MMLV-RT production by IPTG-induction. Bioprocessing techniques are necessary to conduct to obtain higher quality of MMLV-RT under autoinduction system.

Plasmodium falciparum is known to cause severe malaria, current treatment consists in artemisinin-based combination therapy, but resistance can lead to treatment failure. Knowledge concerning P. falciparum essential proteins can be used for searching new antimalarials, among these a potential candidate is shikimate dehydrogenase (SDH), an enzyme part of the shikimate pathway which is responsible for producing endogenous aromatic amino acids. SDH from P. falciparum (PfSDH) is unexplored by the scientific community, therefore, this study aims to establish the first protocol for active PfSDH expression. Putative PfSDH nucleotide sequence was used to construct an optimized expression vector pET28a+PfSDH inserted in E. coli BL21(DE3). As a result, optimal expression conditions were acquired by varying IPTG and temperature through time. Western Blot analysis was applied to verify appropriate PfSDH expression, solubilization and purification started with lysis followed by two-steps IMAC purification. Enzyme activity was measured spectrophotometrically by NADPH oxidation, optimal PfSDH expression occur at 0.1 mM IPTG for 48 hours growing at 37 °C and shaking at 200 rpm, recombinant PfSDH obtained after purification was soluble, pure and its physiological catalysis was confirmed. Thus, this study describes the first protocol for heterologous expression of PfSDH in soluble and active form.

Inherently identical cells exhibit significant phenotypic variation. It can be essential for many biological processes and is known to arise from stochastic, \'noisy\', gene expression that is determined by intrinsic and extrinsic components. It is now obvious that the noise varies as a function of inducer concentration. However, its fluctuation over the cell cycle is limited. Applying dual colour fluorescence protein reporter system, Cyan Fluorescent Protein (CFP) and Yellow fluorescent protein (YFP) tagged multi-copy plasmids, we determine variation of the noise components over the phases in lac promoter induced by Isopropyl β-D-1-thiogalactopyranoside (IPTG) and in presence of additional Magnesium, Mg2+ ion. We, also, estimate the how such system deviates from observations of single-copy plasmid. Found 25 % difference between multi-copy system and single-copy system clarifies that observed noise is considerable and estimates population behaviour during the cell cycle. We show that total variation in cells induced with IPTG is determined by higher extrinsic than intrinsic noise. It increases from Lag to Exponential phase and decreases from Retardation to Stationary phase. By observing slow and fast dividing cells, we show that 5 mM Mg2+ increases population homogeneity compared to 2.5 mM Mg2+ in the environment. The experimental data obtained using dual colour fluorescence protein reporter system demonstrates that protein expression noise, depending on intra cellular ionic concentration, is tightly controlled by phase of the cell.

Vaccinia virus (Orthopoxvirus) F17 protein is a major virion structural phosphoprotein having a molecular weight of 11 kDa. Recently, it was shown that F17 synthesised in infected cells interacts with mTOR subunits to evade cell immunity and stimulate late viral protein synthesis. Several years back, we purified an 11 kDa protein that inhibited protein synthesis in reticulocyte lysate from virions, and that possesses all physico-chemical properties of F17 protein. To investigate this discrepancy, we used defective vaccinia virus particles devoid of the F17 protein (designated iF17- particles) to assess their ability to inhibit protein synthesis. To this aim, we purified iF17- particles from cells infected with a vaccinia virus mutant which expresses F17 only in the presence of IPTG. The SDS-PAGE protein profiles of iF17- particles or derived particles, obtained by solubilisation of the viral membrane, were similar to that of infectious iF17 particles. As expected, the profiles of full iF17- particles and those lacking the viral membrane were missing the 11 kDa F17 band. The iF17- particles did attach to cells and injected their viral DNA into the cytoplasm. Co-infection of the non-permissive BSC40 cells with a modified vaccinia Ankara (MVA) virus, expressing an mCherry protein, and iF17- particles, induced a strong mCherry fluorescence. Altogether, these experiments confirmed that the iF17- particles can inject their content into cells. We measured the rate of protein synthesis as a function of the multiplicity of infection (MOI), in the presence of puromycin as a label. We showed that iF17- particles did not inhibit protein synthesis at high MOI, by contrast to the infectious iF17 mutant. Furthermore, the measured efficiency to inhibit protein synthesis by the iF17 mutant virus generated in the presence of IPTG, was threefold to eightfold lower than that of the wild-type WR virus. The iF17 mutant contained about threefold less F17 protein than wild-type WR. Altogether these results strongly suggest that virion-associated F17 protein is essential to mediate a stoichiometric inhibition of protein synthesis, in contrast to the late synthesised F17. It is possible that this discrepancy is due to different phosphorylation states of the free and virion-associated F17 protein.

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.

DNA methylases of the restriction-modifications (R-M) systems are promising enzymes for the development of novel molecular and synthetic biology tools. Their use in vitro enables the deployment of independent and controlled catalytic reactions. This work aimed to produce recombinant DNA methylases belonging to the R-M systems, capable of in vitro inhibition of the type IIS restriction enzymes BsaI, BpiI, or LguI. Non-switchable methylases are those whose recognition sequences fully overlap the recognition sequences of their associated endonuclease. In switch methylases, the methylase and endonuclease recognition sequences only partially overlap, allowing sequence engineering to alter methylation without altering restriction. In this work, ten methylases from type I and II R-M systems were selected for cloning and expression in E. coli strains tolerant to methylation. Isopropyl β-D-1-thiogalactopyranoside (IPTG) concentrations and post-induction temperatures were tested to optimize the soluble methylases expression, which was achieved with 0.5 mM IPTG at 20 °C. The C-terminal His6-Tag versions showed better expression than the N-terminal tagged versions. DNA methylation was analyzed using purified methylases and custom test plasmids which, after the methylation reactions, were digested using the corresponding associated type IIS endonuclease. The non-switchable methylases M2.Eco31I, M2.BsaI, M2.HpyAII, and M1.MboII along with the switch methylases M.Osp807II and M2.NmeMC58II showed the best activity for site-selective inhibition of type IIS restriction enzyme activity. This work demonstrates that our recombinant methylases were able to block the activity of type IIS endonucleases in vitro, allowing them to be developed as valuable tools in synthetic biology and DNA assembly techniques. KEY POINTS: • Non-switchable methylases always inhibit the relevant type IIS endonuclease activity • Switch methylases inhibit the relevant type IIS endonuclease activity depending on the sequence engineering of their recognition site • Recombinant non-switchable and switch methylases were active in vitro and can be deployed as tools in synthetic biology and DNA assembly.

Clustered regularly interspaced short palindromic repeats (CRISPR) are prokaryotic adaptive immune systems regularly utilized as DNA-editing tools. While Neisseria gonorrhoeae does not have an endogenous CRISPR, the commensal species Neisseria lactamica encodes a functional Type I-C CRISPR-Cas system. We have established an isopropyl β-d-1-thiogalactopyranoside added (IPTG)-inducible, CRISPR interference (CRISPRi) platform based on the N. lactamica Type I-C CRISPR missing the Cas3 nuclease to allow locus-specific transcriptional repression. As proof of principle, we targeted a non-phase-variable version of the opaD gene. We show that CRISPRi can downregulate opaD gene and protein expression, resulting in bacterial inability to stimulate neutrophil oxidative responses and to bind to an N-terminal fragment of CEACAM1. Importantly, we used CRISPRi to effectively knockdown all the transcripts of all 11 opa genes using a five-spacer CRISPR array, allowing control of the entire phase-variable opa family in strain FA1090. We also report that repression is reversible following IPTG removal. Finally, we showed that the Type I-C CRISPRi system can conditionally reduce the expression of two essential genes. This CRISPRi system will allow the interrogation of every Gc gene, essential and non-essential, to study physiology and pathogenesis and aid in antimicrobial development.IMPORTANCEClustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have proven instrumental in genetically manipulating many eukaryotic and prokaryotic organisms. Despite its usefulness, a CRISPR system had yet to be developed for use in Neisseria gonorrhoeae (Gc), a bacterium that is the main etiological agent of gonorrhea infection. Here, we developed a programmable and IPTG-inducible Type I-C CRISPR interference (CRISPRi) system derived from the commensal species Neisseria lactamica as a gene repression system in Gc. As opposed to generating genetic knockouts, the Type I-C CRISPRi system allows us to block transcription of specific genes without generating deletions in the DNA. We explored the properties of this system and found that a minimal spacer array is sufficient for gene repression while also facilitating efficient spacer reprogramming. Importantly, we also show that we can use CRISPRi to knockdown genes that are essential to Gc that cannot normally be knocked out under laboratory settings. Gc encodes ~800 essential genes, many of which have no predicted function. We predict that this Type I-C CRISPRi system can be used to help categorize gene functions and perhaps contribute to the development of novel therapeutics for gonorrhea.