In vivo cloning of PCR product via site-specific recombination in Escherichia coli.

Over the past years, several methods have been developed for gene cloning. Choosing a cloning strategy depends on various factors, among which simplicity and affordability have always been considered. The aim of this study, on the one hand, is to simplify gene cloning by skipping in vitro assembly reactions and, on the other hand, to reduce costs by eliminating relatively expensive materials. We investigated a cloning system using Escherichia coli harboring two plasmids, pLP-AmpR and pScissors-CmR. The pLP-AmpR contains a landing pad (LP) consisting of two genes (λ int and λ gam) that allow the replacement of the transformed linear DNA using site-specific recombination. After the replacement process, the inducible expressing SpCas9 and specific sgRNA from the pScissors-CmR (CRISPR/Cas9) vector leads to the removal of non-recombinant pLP-AmpR plasmids. The function of LP was explored by directly transforming PCR products. The pScissors-CmR plasmid was evaluated for curing three vectors, including the origins of pBR322, p15A, and pSC101. Replacing LP with a PCR product and fast-eradicating pSC101 origin-containing vectors was successful. Recombinant colonies were confirmed following gene replacement and plasmid curing processes. The results made us optimistic that this strategy may potentially be a simple and inexpensive cloning method. KEY POINTS: •The in vivo cloning was performed by replacing the target gene with the landing pad. •Fast eradication of non-recombinant plasmids was possible by adapting key vectors. •This strategy is not dependent on in vitro assembly reactions and expensive materials.

In Silico and Experimental Studies on the Effect of α3 and α5 Deletion on the Biochemical Properties of Bacillus thermocatenulatus Lipase.

To investigate the effect of α3 and α5 helices on the biochemical characterization of Bacillus thermocatenulatus lipase (BTL2), both helices were deleted from native BTL2 lipase. After structural modeling and characterization, the truncated btl2 gene (Δbtl2) was cloned into E. coli BL21 under the control of the T7 promoter. After cultivation and induction of the recombinant bacteria, the Δα3α5 lipase was purified by Ni-NTA column chromatography. Next, the biochemical properties of the Δα3α5 lipase were compared with the previously expressed and purified native lipase. In the presence of the substrate tributyrin (C4), the maximum activity of native and Δα3α5 lipase was 9360 and 5000 U/mg, respectively. The deletion changed the substrate specificity from tributyrin (C4) to tricaprylin (C8) substrate. Native and Δα3α5 lipase showed similar activity patterns at all temperatures and pH values, with the activity of Δα3α5 lipase being approximately 20% lower than native lipase. Triton X100 increased the activity of native and Δα3α5 lipases by 2.1- and 2.5-fold, respectively.

Structure and activity of a novel robust peroxidase from Alkanna frigida cell culture.

Demands for peroxidases (POX)s with diverse physicochemical properties have steadily grown as more applications of POXs are demonstrated. Plants are among the best sources of versatile POXs, and plant biotechnology, as an agricultural hassle-free technology, promises to circumvent the limitations of natural resource exploitation and to address the demands. Following this trend, it was shown that POX production steadily increased during the 31-day subculture of Alkanna frigida (from Boraginaceae) callus on Murashige-Skoog medium containing 2,4-dichlorophenoxyacetic acid (10-6 M) and kinetin (10-5 M). The purified cationic enzyme (POXalf) maintained its optimal activity over pH 4-7 for 2 years. It was resistant to H2O2 high concentrations (IC50 = 543.7 mM) and showed high specific activity in the reaction with phenol (4320.5 AU mg-1 > 20-fold of HRP AU). Furthermore, the specificity constant ratio of guaiacol to phenol indicated a 100 times faster reaction of POXalf with guaiacol. However, in contrast to HRP, it had little effect on diazo derivatives of aniline and meta-diaminobenzene. Based on the resulting primary structure from the tandem mass analysis, the POXalf 3D structure was constructed via homology modelling. Despite the high topological similarity between the HRP and POXalf structures, there were important differences between the active site pockets that could explain the observed differences in the corresponding substrate spectra and the specific activities. Considering the dynamics of POXalf production, its inactivity towards IAA and its high affinity for guaiacol, POXalf may have associated roles with A. frigida cell wall construction and monolignol metabolism.

Studying the Expression Efficiencies of Human Clotting Factor IX Analogs, Rationally-designed for Hyper-glycosylation.

Glyco-engineering has attracted lots of interest in studies dealing with the pharmacokinetics of therapeutic proteins. Based on our previous in-silico studies, two sites were selected in the N-terminal gamma-carboxy glutamic acid-rich (Gla) domain of the human clotting factor IX (hFIX) to add new N-glycosylation sites. Site-directed mutagenesis was employed to conduct K22N and R37N substitutions and introduce new N-glycosylation sites in the mature hFIX. The expression efficiencies of the mutants, in parallel with the wild-type hFIX (hFIXwt), were assessed in suspension adapted Chinese hamster ovary (CHO-s) cells at transcriptional, translational, and post-translational levels. The transcription levels of both N-glycosylation mutants were significantly lower than that of the hFIXwt. In contrast, at the protein level, the two hFIX mutants showed higher expression. The occurrence of hyper-glycosylation was only confirmed in the case of the hFIXR37N mutant, which decreased the clotting activity. The higher expression of the hFIX mutants at protein level was evidenced, which could be attributed to higher protein stability, via omitting certain protease cleavage sites. The coagulation activity decline in the hyper-glycosylated hFIXR37N mutant is probably due to the interference of the new N-glycan with protein-protein interactions in the coagulation cascade.

Optimizing the Expression and Solubilization of an E. coli-Produced Leukemia Inhibitory Factor for Anti-LIF Antibody Production and Use Thereof for Contraception in Mice.

Leukemia inhibitory factor (LIF) is an essential cytokine for blastocyst implantation. This study evaluated the effect of LIF inhibition on the blockage of embryo implantation. A truncated mouse LIF (tmLIF) was designed and expressed in E. coli. The protein expression was optimized using different culture media and inducers. To block pregnancy, the mice were immunized by the purified protein via maternal injection of the protein or in utero injection of the anti-LIF serum. The expression of implantation-relevant genes was quantified in the uterine tissue. The results showed that the protein was expressed in aggregated form in E. coli. The highest yield of protein was produced in the M9 medium. The insoluble protein was completely dissociated by SDS and 2-ME combination, but not by urea. The maternal immunization reduced the number of offspring, but not significantly. Instead, in utero injection of the anti-LIF serum prevented the blastocyst implantation. Gene expression analyses showed decrease of Jam2, Msx1and HB-EGF genes and increase of Muc1 gene as the result of intrauterine administration of the anti-LIF serums. In conclusion, SDS-mediated solubilization of inclusion bodies was compatible with in vivo studies. The intrauterine administration of anti-LIF serum could prevent mouse pregnancy. This indicates that in utero application of LIF antibodies might be used as a contraceptive.

Evaluation of multi-epitope recombinant protein as a candidate for a contraceptive vaccine.

Contraceptive vaccine (CV) is a valuable, non-invasive, and alternative method for purposeful contraception. Sperm antigens are useful targets for producing CVs due to their specialized expression in sperm. In this study, a recombinant protein containing three main sperm epitopes (IZUMO1, SACA3, and PH-20) was designed and evaluated as CV to control fertility in male mice. The chimeric recombinant protein was expressed and purified in E. coli. Male mice were immunized by 100 µg purified protein and sera were collected to assess IgG antibodies. Evaluating the reproductive performance, immunized male mice mated with normal-fertile female mice and mating rate and the number of newborns was studied. Immunized mice were sacrificed and necropsy and histopathology studies were conducted. The results revealed that the designed chimeric protein stimulated the immune system of the mice effectively. The level of IgG antibody was significantly higher in vaccinated mouse rather than control mouse. Eighty percent of the vaccinated mice became infertile and in the remaining ones, the number of children decreased to 4-6 offspring instead of 10-12 in normal mice. Histopathological studies showed that no organs including heart, brain, lung, liver, kidney and intestine were damaged. However, Normal spermatogenesis has been disrupted and necrotic spermatogonia cells were reported in Seminiferous tubules. We concluded that the designed chimeric protein containing IZUMO1, SACA3, and PH-20 epitopes can stimulate the immune system and cause male contraception without any side effects.

Cohnella sp. A01 laccase: thermostable, detergent resistant, anti-environmental and industrial pollutants enzyme.

Laccase (EC 1.10.3.2; benzenediol; oxygen oxidoreductases) is a multi-copper oxidase that catalyzes the oxidation of phenols, polyphenols, aromatic amines, and different non-phenolic substrates with concomitant reduction of O2 to H2O. Enzymatic oxidation techniques have the potential of implementation in different areas of industrial fields. In this study, the Cohnella sp. A01 laccase gene was cloned into pET-26 (b+) vector and was transformed to E. coli BL21. Then it was purified using His tag affinity (Ni sepharose resin) chromatography. The estimated molecular weight was approximately 60 kDa using SDS-PAGE. The highest enzyme activity and best pH for 2,6-dimethoxyphenol (DMP) oxidation were recorded as 8 at 90 °C respectively. The calculated half-life and kinetic values including Km, Vmax, turn over number (kcat), and catalytic efficiency (kcat/Km) of the enzyme were 106 min at 90 °C and 686 µM, 10.69 U/ml, 20.3 S-, and 0.029 s-1 µM-1, respectively. The DMP was available as the substrate in all the calculations. Enzyme activity enhanced in the presence of Cu2+, NaCl, SDS, n-hexane, Triton X-100, tween 20, and tween 80, significantly. The binding residues were predicted and mapped upon the modeled tertiary structure of identified laccase. The remaining activity and structural properties of Cohnella sp. A01 laccase in extreme conditions such as high temperatures and presence of metals, detergents, and organic solvents suggest the potential of this enzyme in biotechnological and industrial applications. This process has been patented in Iranian Intellectual Property Centre under License No: 91325.

Bottom-up synthesis of nitrogen and oxygen co-decorated carbon quantum dots with enhanced DNA plasmid expression.

In this paper, a bottom-up hydrothermal route is reported for the synthesis of oxygen and nitrogen co-decorated carbon quantum dots (CQDs) using ammonium hydrogen citrate (AHC) as a single precursor. DLS data approved the formation of 4.0 nm (average size) CQDs. XRD pattern shows the interlayer spacing (002) of 3.5 Šfor CQDs, which is exactly the same as that of crystalline graphite. XPS and FTIR spectra verified the formation of oxygen and nitrogen functional groups on the CQDs surface. Co-decorated carboxyl, hydroxyl and amine groups on the CQDs surfaces make them as promising polyelectrolyte for gene delivery. Toxicity assay showed a survival rate of 70% under different incubation times and up to 500 µg/mL. The highly water-soluble, stable fluorescence and low toxic CQDs increased the gene expression of DNA plasmid in E. coli bacteria 4-fold more than the control group.

An Efficient Approach for Two Distal Point Site-Directed Mutagenesis from Randomly Ligated PCR Products.

Site-directed mutagenesis is one of the most important tools in molecular biology. The majority of the mutagenesis methods have been developed to mutate one region of target DNA in each cycle of mutagenesis, while in some cases there is a need to mutate several distal points. We used a new method to simultaneously mutate two distal points in the target DNA. Different regions of the target DNA were amplified in three separate PCR reactions. The PCR products were back-to-back and together they made the complete length of the template DNA. Mutations were introduced to PCR products by middle mutagenic primers. PCR products were mixed and ligated with random blunt ligation, and then the desired mutated DNA fragments were selected in two steps by flanking restriction enzyme digestion and size selection. Selected fragments were amplified in another PCR reaction using flanking primers and finally cloned into the plasmid vector. This mutagenesis process is simple, there is no need to use modified primers and long or difficult PCR reactions.

In silico and experimental improvement of bacteriorhodopsin production in Halobacterium salinarum R1 by increasing DNA-binding affinity of Bat through Q661R/Q665R substitutions in HTH motif.

DNA-binding motif of bacterioopsin activator (Bat) protein is a Helix-Turn-Helix motif, which binds to bop promoter and induces bacterioopsin (Bop) expression under light and low oxygen tension. Bacterioopsin is linked to retinal to produce bacteriorhodopsin (BR), which in turn supplies energy source in Halobacterium salinarum. In this study, effect of Bat HTH motif-promoter DNA interaction on bacterioopsin (Bop) expression was investigated using in silico and experimental approaches. Molecular docking showed that the most stable DNA-protein complex was generated by Q661R/Q665R mutant. Based on the in silico analysis, HTH motif was mutated using site-directed mutagenesis and Hbt. salinarum recombinant strains were developed by introduction of mutant bat genes. Double positively charged amino acid substitutions (Q661R/Q665R) in second helix of HTH motif increased whereas deletion of this region decreased BR production. However, other single substitutions (Q665R and Q661H) did not change BR production. These findings represent key role of HTH motif stability for DNA binding and regulation of bacterioopsin (Bop) expression and bacteriorhodopsin (BR) production independent of environmental condition.

Rational engineering of Cel5E from Clostridium thermocellum to improve its thermal stability and catalytic activity.

The celH gene from Clostridium thermocellum encodes a protein containing 900 residues and three components, including Cel5E, Lic26a, and carbohydrate-binding domains. Cel5E is a member of the glycoside hydrolase-5 family and is a bifunctional xylanase/cellulase enzyme. We targeted a semi-hydrophobic pocket near the Cel5E active site and theoretically screened mutated variants for enhanced levels of thermal stability. Cel5E mutations were inserted into celH by overlapping polymerase chain reaction, followed by expression of wild-type and mutant enzymes in Escherichia coli BL21 (DE3) and purification by affinity chromatography. Thermal-stabilizing mutations were subjected to molecular dynamics simulation, and measurement of the in vacuo potential energy, van der Waals forces, electrostatic interactions, and net nonbonded potential energies obtained an overall binding affinity of - 64.964 KJ/mol for wild-type Cel5E and - 176.148, - 200.921, and - 120.038 KJ/mol for the N94F, N94W, and E133F mutants, respectively. Additionally, the N94W, N94F, E133F, and N94A variants exhibited 1.92-, 1.29-, 1.1-, and 1.15-fold better carboxymethyl cellulase (CMCase) and 1.46-, 1.29-, 1.11-, and 1.12-fold better ß-glucanase activity on barley ß-glucan relative to the wild-type enzyme. Interestingly, the optimal temperature for CMCase activity by the N94W variant was shifted 2 °C higher than that for the wild-type enzyme. Mutated variants showed improved CMCase and ß-glucanase activity and shifted toward higher temperature of maximum activity.

Practical evaluation of 11 de novo assemblers in metagenome assembly.

Next Generation Sequencing (NGS) technologies are revolutionizing the field of biology and metagenomic-based research. Since the volume of metagenomic data is typically very large, De novo metagenomic assembly can be effectively used to reduce the total amount of data and enhance quality of downstream analysis, such as annotation and binning. Although, there are many freely available assemblers, but selecting one suitable for a specific goal can be highly challenging. In this study, the performance of 11 well-known assemblers was evaluated in the assembly of three different metagenomes. The results obtained show that metaSPAdes is the best assembler and Megahit is a good choice for conservative assembly strategy. In addition, this research provides useful information regarding the pros and cons of each assembler and the effect of read length on assembly, thereby helping scholars to select the optimal assembler based on their objectives.

Improving the thermostability of Serratia marcescens B4A chitinase via G191V site-directed mutagenesis.

Chitinases with high thermostability are important for many industrial and biotechnological applications. This study was conducted to enhance the stability of Serratia marcescens B4A chitinase by site directed mutagenesis of G191 V. Further characterization showed that the thermal stability of the mutant showed marked increase of about 5 and 15 fold at 50 and 60 °C respectively, while the optimum temperature and pH was retained. Kinetic analysis showed decreased Km and Vmax of the mutant in comparison with the wild type chitinase of about 1.3 and 3 fold, respectively. Based on structural prediction, it was speculated that this replacement shortened an important loop concomitant with the extension of adjacent ß sheets. Accordingly, a higher thermostability of G191 V up to 90 °C supporting the decreased flexibility of unfolded state was also indicated. Finally, a practical proof of kinetic and thermal stabilization of chitinase was provided through decreased flexibility and entropic stabilization of its surface loops.

Biochemical Characterization of Recombinant Thermostable Cohnella sp. A01 ß-Glucanase

Background: Typically, non-cellulytic glucanase, including fungi and yeast cell wall hydrolyzing enzymes, are released by some symbiotic fungi and plants during the mycoparasitic fungi attack on plants. These enzymes are known as the defense mechanisms of plants. This study intends to investigate the biochemical properties of ß-1,6-glucanase (bg16M) from native thermophilic bacteria, Cohnella A01. Methods: bg16M gene was cloned and expressed in E. coli BL21 (DE3). The enzyme was purified utilizing Ni-NTA nikcle sepharose column. Pustulan and laminarin were selected as substrates in enzyme assay. The purified bg16M enzyme was treated with different pH, temperature, metal ions, and detergents. Results: The expressed protein, including 639 amino acids, showed a high similarity with the hydrolytic glycosylated family 30. The molecular weight of enzyme was 64 kDa, and purification yield was 46%. The bg16M demonstrated activity as 4.83 U/ml on laminarin and 2.88 U/ml on pustulan. The optimum pH and temperature of the enzyme were 8 and 50 °C, respectively. The enzyme had an appropriate stability at high temperatures and in the pH range of 7 to 9, showing acceptable stability, while it did not lose enzymatic activity completely at acidic or basic pH. None of the studied metal ions and chemical compounds was the activator of bg16M, and urea, SDS, and copper acted as enzyme inhibitors. Conclusion: Biochemical characterization of this enzyme revealed that bg16M can be applied in beverage industries and medical sectors because of its high activity, as well as thermal and alkaline stability.

Molecular Engineering of the Geobacillus stearothermophilus α-Amylase and Cel5E from Chlostridium thermocellim; In Silico Approach.

BACKGROUND: Considering natural thermal stability, Geobacillus stearothermophilus amylase and Cel5E from Clostridium thermocellum are good candidates for industrial applications. To be compatible with the industrial applications, this enzyme should be stable in the high temperatures, so any improvement in their thermal stability is valuable. OBJECTIVES: Using in silico approach and identifying point mutations in the structure amylase of G. stearothermophilus and Cel5E from C. termocellum we tried to increase thermal stability of the enzymes along with their catalytic activity to reach a new industrial amylase with higher thermostability and an improved function. MATERIALS AND METHODS: In this study we predicted the 3D structure of the enzymes, then simulated the molecular docking study using MolDock, PLANTS, and Lamarkian genetic algorithm as scoring functions for the docking and in silico engineering of the protein aiming to increase the thermal stability and catalytic activity. RESULTS: A series of thermal stability increasing point mutations were exerted around the active site of the enzyme, then by docking procedure, the binding affinity was measured and finally a list of mutations which theoretically improved the increased thermal stability as well as catalytic activity were proposed. CONCLUSIONS: Based on the in silico results obtained the modified enzymes seems to be suitable candidates for considering in both laboratory and industrial scales.

Role of Q177A and K173A/Q177A substitutions in thermostability and activity of the ELBn12 lipase.

Thermostable lipases have many applications in detergent industries and in organic synthesis. There are many ways to improve thermal stability of enzymes, for example, higher hydrophobicity, greater structural packing, higher content of the charged residues, and lower thermolabile ones. In this study, thermolabile Gln (sensitive to higher temperatures) was substituted with Ala in native ELBn12 and mutated K173A lipases to examine its effect on thermal stability and activity of the lipases. Single (Q177A) and double mutants (K173A/Q177A) were expressed in Escherichia coli pLysS. The Q177A variant increased both activity and thermostability of the lipase, whereas K173A/Q177A had a negative effect on the lipase activity and only had better thermal stability than the native at 50 °C. pH stability of the double mutant between 9.0 and 11 was also lower than the other variants. Stability analysis in the presence of chemicals showed that Q177A mutant had better activity with 50% (v/v) organic solvents. On the other hand, K173A lipase showed increased activity with 1% (w/v) nonionic surfactant, and finally K173A/Q177A had better stability with 10 mM metal ions compared to the native lipase.

Evaluation of nine popular de novo assemblers in microbial genome assembly.

Next generation sequencing (NGS) technologies are revolutionizing biology, with Illumina being the most popular NGS platform. Short read assembly is a critical part of most genome studies using NGS. Hence, in this study, the performance of nine well-known assemblers was evaluated in the assembly of seven different microbial genomes. Effect of different read coverage and k-mer parameters on the quality of the assembly were also evaluated on both simulated and actual read datasets. Our results show that the performance of assemblers on real and simulated datasets could be significantly different, mainly because of coverage bias. According to outputs on actual read datasets, for all studied read coverages (of 7×, 25× and 100×), SPAdes and IDBA-UD clearly outperformed other assemblers based on NGA50 and accuracy metrics. Velvet is the most conservative assembler with the lowest NGA50 and error rate.

Production and Characterization of Monoclonal Antibody against Recombinant Virus Coat Protein CP42.

There are many studies related to the production of a ELISA kit for diagnosing virus infections. However, production of most kits depends on purification of whole virus particles, which involves the use of costly equipment and reagents. The purpose of this study was to check out if the anti-CP42 antibodies could be used as a diagnostic assay for detection of Grapevine fanleaf Virus (GFLV). In this study, recombinant GFLV coat protein gene related to selected antigenic determinants was inserted into pET-28a bacterial expression vector and the construct (pET-28a CP42) was cloned into E. coli strain (DE3). Expressed protein was verified with western blotting assay by the use of commercially available anti-GFLV antibody. The recombinant protein was purified using nickel-nitrilotriacetic acid (Ni-NTA) resin. Balb/c mice were immunized with purified protein and splenocytes of hyperimmunized mice were fused with murine myeloma Sp2/0 cells. Positive hybridomas were selected by ELISA using CP42 as coating antigen. The results showed that monoclonal antibody (MAb) specific to CP42 has been successfully generated. Efficiency of produced antibody was analyzed by ELISA and western blotting assay using some confirmed grapevine samples. The infection was confirmed previously based on morphological features and ELISA assay, performed using commercial anti-GFLV antibody. The monoclonal antibody reacted with antigen in ELISA and immunoblot method. Our results demonstrated that anti recombinant CP42 monoclonal antibodies are able to diagnose whole virus in infected grapevine sample using ELISA test.

The importance of the non-active site and non-periodical structure located histidine residue respect to the structure and function of exo-inulinase.

Here, we have studied the role of a histidine residue with the lowest solvent accessibility among other histidine residues at the end of a short connecting structure (189AELH192) of the catalytic domain of the exo-inulinase through creation of H192A mutant. Site-directed mutagenesis method was applied to create the mutant enzyme. Molecular dynamics (MD) simulations, spectroscopic, calorimetric and kinetics analysis were used to study the structural and functional consequences of His192 substitution. Accordingly, the thermo-stabilities and catalytic performance were decreased upon H192A mutation. In silico and experimental approaches evidently confirm that His192 residue of exo-inulinase possesses structural and functional importance regardless of the lack of direct interaction with the substrate or involvement in the catalytic activity of exo-inulinase.

Thermostable chitinase from Cohnella sp. A01: isolation and product optimization

Abstract Twelve bacterial strains isolated from shrimp farming ponds were screened for their growth activity on chitin as the sole carbon source. The highly chitinolytic bacterial strain was detected by qualitative cup plate assay and tentatively identified to be Cohnella sp. A01 based on 16S rDNA sequencing and by matching the key morphological, physiological, and biochemical characteristics. The cultivation of Cohnella sp. A01 in the suitable liquid medium resulted in the production of high levels of enzyme. The colloidal chitin, peptone, and K2HPO4 represented the best carbon, nitrogen, and phosphorus sources, respectively. Enzyme production by Cohnella sp. A01 was optimized by the Taguchi method. Our results demonstrated that inoculation amount and temperature of incubation were the most significant factors influencing chitinase production. From the tested values, the best pH/temperature was obtained at pH 5 and 70 °C, with Km and V max values of chitinase to be 5.6 mg/mL and 0.87 µmol/min, respectively. Ag+, Co2+, iodoacetamide, and iodoacetic acid inhibited the enzyme activity, whereas Mn2+, Cu2+, Tweens (20 and 80), Triton X-100, and EDTA increased the same. In addition, the study of the morphological alteration of chitin treated by enzyme by SEM revealed cracks and pores on the chitin surface, indicating a potential application of this enzyme in several industries.