Strain DH5α Research Articles

Agrobacterium tumefaciens -mediated transformation of tomato (Solanum lycopersicum)

The present investigation describes the development of genetically engineered tomato plants with annexin gene. The alkaline lysis method is used to isolate the plasmid DNA of pUC 19 vector having the desired Annexin gene (3Kbp) and the plasmid DNA of the binary vector pGPTV (13 Kbp) from E.coli (DH5α strain). The purified pUC 19/Annexin and pGPTV plasmid were restriction digested using the restriction enzymes EcoRI and XbaI as a linearised band was eluted from the gel. The digested plasmid shown in the band pattern in the gel were cut by gel elution technique and purified from other reaction mixture. Then the dot spot test was done to calculate the concentration of pGPTV and Annexin gene. The recombinant PGPTV plasmid with the annexin gene in Agrobacterium tumefaciens MTCC 431 was mobilized and transferred to plant system through the mobilization helper plasmid pRK2013. The kanamycin resistance gene (NPT II) was used as a selective marker. The calli used for isolating the genomic DNA which was then amplified for confirmation of annexin gene. The nptII gene of 800 bp serves as a selectable marker system in plants and its amplification confirmed the presence of annexin gene in transgenic plants by PCR method.

Design, cloning and expression assay of oipA gene in a bicistronic vector harboring mice IL-18 gene: potential implications for Helicobacter pylori vaccine investigations

Introduction: Helicobacter pylori (H. pylori) infection has remained as a global health problem. Animal studies demonstrated the role of H. pylori oipA gene in the development of gastric cancer. The aim of this study was the cloning and expression of Helicobacter pylori oipA gene in a bicistronic vector harboring mice IL-18 gene. Materials and methods: The target gene encoding oipA was amplified from a codonoptimized clone by PCR, and then double-digested by restriction enzymes. The pIRESIgk/ mIL18/Fc plasmid was simultaneously digested by BstXI/NotI enzymes to elicit the eGFP segment. PCR product of oipA was inserted into pIRES-Igk/mIL18/Fc plasmid using T4 ligase. Transformation into DH5α strain was done. Cloning was confirmed by PCR, enzymatic digestion and sequencing. Expression of the oipA and IL-18 mRNA was assessed by means of TaqMan Real-time PCR. Results: Electrophoresis of PCR product, enzymatic digestion and sequencing showed that the H. pylori oipA gene was successfully cloned into pIRES-Igk/mIL18/Fc to generate mIL- 18-pIRES2-oipA plasmid. The results of Real-time PCR confirmed the successful expression of both oipA and IL-18 in mouse macrophage cell line. Conclusion: Considering the role of oipA in pathogenesis of H. pylori and potent activity of IL-18 as a molecular adjuvant, the results of the present study showed that the expression of codon-optimized oipA gene in bicistronic vector including mouse IL-18 is successful. So, it could be considered as an appropriate genetic vaccine candidate for H. pylori in future investigations.

Agrobacterium tumefaciens -mediated transformation of tobacco (Nicotiana tabaccum)

The present study involves the development of genetically engineered tobacco plants with annexin gene. The plasmid DNA of pUC 19 vector having the desired Annexin gene (3Kbp) and the plasmid DNA of the binary vector pGPTV (13 Kbp) were isolated from E.coli (DH5α strain) by alkaline lysis method. The purified pUC 19/Annexin and pGPTV plasmid were restriction digested using the restriction enzymes EcoRI and XbaI as a linearised band was eluted from the gel. The digested plasmid shown in the band pattern in the gel were cut by gel elution technique and purified from other reaction mixture. Then the dot spot test was done to calculate the concentration of pGPTV and Annexin gene. The recombinant PGPTV plasmid with the annexin gene in Agrobacterium tumefaciens MTCC 431 was mobilized and transferred to plant system through the mobilization helper plasmid pRK2013. The kanamycin resistance gene (NPT II) was used as a selective marker. The calli used for isolating the genomic DNA which was then amplified for confirmation of annexin gene. The nptII gene of 800 bp serves as a selectable marker system in plants and its amplification confirmed the presence of annexin gene in transgenic plants by PCR method.

On-Chip Oval-Shaped Nanocavity Photonic Crystal Waveguide Biosensor for Detection of Foodborne Pathogens

A photonic crystal waveguide (PCW) biosensor is proposed for the detection of foodborne pathogens. Various semiconductor materials and insulator with higher to lower refractive indices (Si, GaAs, Si3N4, and SiO2) are analyzed to fix the choice of material in PCW design. The design and analysis are performed using finite difference time domain (FDTD) simulation method. The design exhibits two inverted J-shaped defects with center cavity designed in the shape of Escherichia coli. In this research, DH5α strain of E. coli foodborne pathogens is considered as a model due to its shape. Simulation of PCW design is performed using infrared radiation (1 and 1.55 μm) wavelengths. Simulation analysis reports larger resonance wavelength shifts, higher sensitivities, and quality factors for Si-based PCW biosensor at an operating wavelength of 1.55 μm.

Antimicrobial activity of eumelanin-based hybrids: The role of TiO2 in modulating the structure and biological performance

Eco-friendly hybrid Eumelanin-TiO2 nanostructures, recently obtained through in situ methodology based on hydrothermal route, have shown a striking antimicrobial activity, after exposure to oxidative environment, even under visible light induction condition. Nevertheless, the role of each component in defining the efficacy of these biological properties is far from being clearly defined. Furthermore, the effect of oxidative step on hybrids structure has not yet addressed. This study aims at elucidating the role of the ratio between eumelanin precursor, 5,6-dihydroxyindole-2-carboxylic acid (DHICA), and TiO2, for its polymerization in defining morphology and structural organization of TiO2-melanin nanostructures.Furthermore, tests on a Gram-negative Escherichia coli DH5α strain under UV irradiation and even visible light allowed to assess the contribution of each component, as well as of the TiO2–DHICA charge transfer complex to overall biological performance. Finally, results of biocide characterization were combined with spectroscopic evidences to prove that oxidative treatment induces a marked structural modification in melanin thus enhancing overall antimicrobial efficacy.

Self-Conjugation of the Enteropathogenic Escherichia coli Adherence Factor Plasmid of Four Typical EPEC Isolates.

The enteropathogenic Escherichia coli (EPEC) adherence factor plasmid (pEAF) encodes the proteins involved in the biogenesis of the bundle-forming pilus (BFP), a key virulence factor that mediates microcolony formation and the localized adherence phenotype on the surface of the host enterocytes. The presence or absence of this plasmid defines typical EPEC (tEPEC) and atypical EPEC (aEPEC), respectively. Although lateral transfer of pEAF has been evidenced by phylogenetic studies, conjugal transfer ability has been experimentally established only for two pEAF plasmids from strains isolated in the late 60s. In the present work, we tested the self-conjugation ability of four pEAF plasmids from tEPEC strains isolated between 2007 and 2008 from children in Peru and the potential of aEPEC to receive them. A kanamycin resistance cassette was inserted into donor pEAF plasmids in order to provide a selectable marker in the conjugation experiments. Two aEPEC isolated from the same geographic region were used as recipient strains along with the laboratory E. coli DH5α strain. Here we show that the four pEAF plasmids tested are self-conjugative, with transfer frequencies in the range of 10−6 to 10−9. Moreover, the generation of aEPEC strains harboring pEAF plasmids provides valuable specimens to further perform functional studies.

Genetic stability of an Escherichia coli strain engineered to produce a novel therapeutic DNA vaccine encoding chicken type II collagen for rheumatoid arthritis

Abstract The development of therapeutic DNA vaccines capable of recovering immunological tolerance through the induction of both CD4 + CD25 + FoxP3 + regulatory and CD3 + CD8 + C28-suppressor T cells, and/or inhibition of both autoreactive CD4 + CD28+ type 1 T helper and autoantibody-producing B cells offers a promising new strategy for the treatment of rheumatoid arthritis. Previously, we developed pcDNA-CCOL2A1, a novel therapeutic DNA vaccine, which encodes the full-length chicken type II collagen sequence, and demonstrated that the efficacy of this vaccine for treating rheumatoid arthritis was comparable to that of the current “gold standard” treatment, methotrexate. In this study, we investigated the genetic stability of a strain engineered to produce the vaccine during continuous passage and long-term storage at different temperatures. By screening a panel of 12 strains, we identified a DH5α strain that exhibited high levels (12.30 ± 0.05 mg L−1) of pcDNA-CCOL2A1 production after 15 h cultivation, and subsequently utilized this strain to establish a three-tier cells bank for future studies. Continuous passage of this strain for 100 inoculation times demonstrated that a higher percentage (>95%) of cells maintained the plasmid when cultivated under selective pressure (ampicillin) than under nonselective conditions, suggesting that the presence of antibiotics in the medium prevents the loss of the pcDNA-CCOL2A1 plasmid. Meanwhile, restriction digestion and gene sequencing analyses demonstrated that the pcDNA-CCOL2A1 vector remained stable, and that the plasmid sequence was conserved during this period. Lastly, the DH5α pcDNA-CCOL2A1 strain exhibited a high plasmid preservation (>90%) and high levels of plasmid production (9.05mg L−1) after storage for 60 months at −80 °C. Furthermore the plasmid extracted from the DH5α pcDNA-CCOL2A1 strain after storage for 60 months at −80 °C was transfected to COS-7 cells, it can stably express the target protein chicken type II collagen. Conversely, this strain exhibited a complete loss of capability after 24 and 18 months storage at −20 °C and 4 °C, respectively. These findings will facilitate further pilot-scale testing, and even industrial-scale production, of the novel therapeutic vaccine pcDNA-CCOL2A1.

Improved System for Constructing Bacterial cDNA Libraries From the Venom Glands of Two Iranian Scorpions

Background: Analyzing a cell or a tissue transcriptome is the best way to understand its functions. cDNA library construction is one of the best methods to achieve this aim, and it is used to determine the structure, functions, and expression profile of a transcriptome. In the construction of a cDNA library, bacterial hosts are very adaptive. One of the most adaptive bacterial hosts is the DH5α strain of Escherichia coli. Objectives: The venom peptides of scorpions are applicable in biological and medical research as potent drugs, as they have bioactive components; therefore, their identification is very important. In this project, we set up and improved the Clontech® In-Fusion SMARTer™ Directional cDNA library construction kit in the transcriptome analysis of the venom glands of two Iranian scorpions, Odonthubuthus doriae (O. doriae) and Mesobuthus eupeus (M. eupeus). Methods: RNA extraction and cDNA synthesis and purification were conducted on the venom glands of O. doriae and M. eupeus scorpions. cDNA libraries were constructed by cloning and transforming chemically competent bacterial cells. The blue-white screen method was used to identify the transformed cells. For finding colonies that have scorpion-related inserts, a colony PCR method was designed with specific primers for two sides of the insertion site into the vector. Results: The obtained white colonies have vectors that include cDNA related to O. doriae and M. eupeus. Conclusions: The constructed cDNA libraries of O. doriae and M. eupeus glands confirmed that a framework was created for the transcriptome analysis and peptide study of the venom glands of other scorpion species.

Active layer identification of photonic crystal waveguide biosensor chip for the detection ofEscherichia coli

This work represents experimental and simulation analysis of photonic crystal waveguide (PCW)-based biosensor structures, which is used for detection of the Escherichia coli (E. coli) cell. A method is adopted for E. coli culture to measure length, diameter, and refractive index to finalize the structural design and to verify the suitability of PCW as a biosensor. This method is tested using DH5α strains of E. coli. The typical precisions of measurements are varied in ranges from 1.132 to 1.825 μm and from 0.447 to 0.66 μm for pathogen’s length and diameter, respectively. The measured distribution of samples over length and diameter are in correlation with the measurements performed by scanning electron microscope. After obtaining average length and diameter of cylindrical shaped E. coli cell, we consider these values for simulation analysis of designed PCW biosensor. E. coli cell is trapped in the middle of the PCW biosensor having three different types of waveguides, i.e., gallium arsenide/silicon dioxide (GaAs/SiO2), silicon/silicon dioxide (Si/SiO2), or silicon nitride/silicon dioxide (Si3N4/SiO2) to observe the maximum resonance shift and sensitivity. It is observed from the simulation data analysis that GaAs/SiO2 is the preferred PCW biosensor for the identification of E. coli.

Microbial metabolism of thiopurines: A method to measure thioguanine nucleotides

Thiopurines are anti-inflammatory prodrugs. We hypothesised that bacteria may contribute to conversion to active drug. Escherichia coli strain DH5α was evaluated to determine whether it could metabolise the thiopurine drugs, thioguanine or mercaptopurine, to thioguanine nucleotides. A rapid and reliable high performance liquid chromatography (ultraviolet detection) method was developed to quantify indirectly thioguanine nucleotides, by measuring thioguanine nucleoside.

Rapidly Probing Antibacterial Activity of Graphene Oxide by Mass Spectrometry-based Metabolite Fingerprinting.

Application of nanomaterials as anti-bacteria agents has aroused great attention. To investigate the antibacterial activity and antibacterial mechanism of nanomaterials from a molecular perspective is important for efficient developing of nanomaterial antibiotics. In the current work, a new mass spectrometry-based method was established to investigate the bacterial cytotoxicity of graphene oxide (GO) by the metabolite fingerprinting of microbes. The mass spectra of extracted metabolites from two strains DH5α and ATCC25922 were obtained before and after the incubation with nanomaterials respectively. Then principal component analysis (PCA) of these spectra was performed to reveal the relationship between the metabolism disorder of microbes and bactericidal activity of GO. A parameter “D” obtained from PCA scores was proposed that is capable to quantitatively evaluate the antibacterial activity of GO in concentration and time-dependent experiments. Further annotation of the fingerprinting spectra shows the variabilities of important metabolites such as phosphatidylethanolamine, phosphatidylglycerol and glutathione. This metabolic perturbation of E. coli indicates cell membrane destruction and oxidative stress mechanisms for anti-bacteria activity of graphene oxide. It is anticipated that this mass spectrometry-based metabolite fingerprinting method will be applicable to other antibacterial nanomaterials and provide more clues as to their antibacterial mechanism at molecular level.

Hydraphiles enhance antimicrobial potency against Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis

Hydraphiles are synthetic amphiphiles that form ion-conducting pores in liposomal membranes. These pores exhibit open-close behavior when studied by planar bilayer conductance techniques. In previous work, we showed that when co-administered with various antibiotics to the DH5α strain of Escherichia coli, they enhanced the drug’s potency. We report here potency enhancements at low concentrations of hydraphiles for the structurally and mechanistically unrelated antibiotics erythromycin, kanamycin, rifampicin, and tetracycline against Gram negative E. coli (DH5α and K-12) and Pseudomonas aeruginosa, as well as Gram positive Bacillus subtilis. Earlier work suggested that potency increases correlated to ion transport function. The data presented here comport with the function of hydraphiles to enhance membrane permeability in addition to, or instead of, their known function as ion conductors.

Surface characterization and biological evaluation of silver-incorporated DLC coatings fabricated by hybrid RF PACVD/MS method

Since the biological response of the body towards an implanted material is mainly governed by its surface properties, biomaterials are improved by various kinds of coatings. Their role is to provide good mechanical and biological characteristics, and exclude some disadvantages like post-implantation infections. This phenomenon may be reduced by introduction of silver as an antibacterial agent. This study evaluates the Ag-DLC films synthesized by the hybrid RF PACVD/MS method according to the patent number PL401955-A1 worked out inter alia by the authors. Such tests as XPS, SEM, EDS, AFM, FTIR, Raman and ICP-TOF-MS were used to determine surface properties of the coatings. The obtained results were correlated with the biological response estimated on the basis of cells viability assay (osteoblast cells line Saos-2) and bacterial colonization test (Escherichia coli strain DH5α). Results showed that the hybrid RF PACVD/MS method allows one to get tight coating preventing the diffusion of harmful elements from the metallic substrate. Ag concentration increases with the growing power density, it occurs in metallic state, does not create chemical bonds and is evenly dispersed within the DLC matrix in the form of nanoscale silver clusters. Increasing silver content above 2at.% improves bactericidal properties, but decreases cell viability

Characterization and Evaluation of HepDOCA-doxorubicin Tagged Magnetic Nanoparticles for Hyperthermia

This paper reports the hyperemia effect studies of drug-magnetic iron oxide nanoparticle conjugates (MION) for targeted cancer therapy. MIONs were synthesized by the method of co-precipitation using salts of iron as precursors. Chemically modified heparin derivative (heparin deoxycholate, hepDOCA) was complexed to chemotherapeutic drug, doxorubicin (DOX) and tagged to MIONs, which act as the drug-vectors. Each of these conjugates was characterized by SEM and DLS. To observe the effect of formulations, an inductor RF coil circuit was set up which ensured that the MIONs heat up in an AC magnetic field of 0.79 mT at 960 kHz. Hyperthermia treatment was performed on E.coli (DH5-α strain) cells and the growth of bacteria along with the protein released upon cell lysis was monitored. It was confirmed that HepDOCA- DOX-MION complex imparts damage to E.coli cells by the magneto-thermo-cytolysis on application of alternating magnetic field.

Comparative analysis of the Shiga toxin converting bacteriophage first detected in Shigella sonnei

Here we report the first complete nucleotide sequence of a Shiga toxin (Stx) converting phage from a Shigella sonnei clinical isolate that harbors stx1 operon, first identified in the chromosome of Shigella dysenteriae type 1. The phage named Shigella phage 75/02 Stx displayed Podoviridae morphology. It proved to be transferable to Escherichia coli K-12 strains, and cytotoxicity of the lysogenized strains was demonstrated in Vero cell cultures. Genomic analysis revealed that the prophage genome is circular and its size is 60,875nt that corresponds to 76 ORFs. The genome of Shigella phage 75/02 Stx shows a great degree of mosaic structure and its architecture is related to lambdoid phages. All the deduced proteins, including the 37 hypothetical proteins showed significant homologies to Stx phage proteins present in databases. The phage uniformly inserted into the ynfG oxidoreductase gene framed by phage integrase and antirepressor genes in parental S. sonnei and in the three lysogenized K-12 strains C600, DH5α and MG1655. The Stx1 prophage proved to be stable in its bacterial hosts and remained inducible.

Volatiles and seasonal variation of the essential oil composition from the leaves of Clinopodium macrostemum var. laevigatum and its biological activities

Clinopodium macrostemum var. laevigatum is a Mexican medicinal plant appreciated because of the pleasant smell of its leaves. This work describes the seasonal volatile content of the leaves by SPME technique as well as the GC–MS analyses of four seasonal essential oils. Menthone (∼34%) and piperitone oxide (∼30%) were the most abundant compounds in all the seasons studied. Antioxidant activity was determined in the seasonal essential oils by DPPH and β-carotene bleaching methods revealing an IC50 range of 0.92–1.46 and 1.30–1.94gL−1, respectively. The enzymatic assays with α-glucosidase and porcine pancreatic lipase showed a non-competitive inhibition caused by the essential oil of this plant. Seasonal IC50 range was 0.14–0.29gL−1 for α-glucosidase and 0.12–0.34gL−1 for porcine pancreatic lipase. The enzymatic assays with acetylcholinesterase demonstrated that the essential oil produced a competitive inhibition with a seasonal IC50 range of 0.17–0.43gL−1. The MIC’s in gL−1 of the essential oil demonstrated a remarkable antimicrobial activity on Erwinia carotovora (0.145), Agrobacterium tumefaciens (0.149), Clavibacter michiganensis (0.184), Pseudomonas syringae pv. phaseolitica (0.381), Pseudomonas syringae pv. glycinea (0.437), Escherichia coli strain DH5α (0.515), Fusarium oxysporum (2.3), Aspergillus niger (2.9) and Rhizopus stolonifer (3.6). The antimicrobial activity of the essential oil was maintained in the seasons studied. The described properties of the essential oil of this traditional crop could probably be used to design new products with industrial application.

Molecular characterisation of Bacillus chitinase for bioconversion of chitin waste

In this work chitin was extracted chemically from shrimp shells. Seventeen Bacillus isolates were screened for chitinolytic activity. The chitinolytic strains of Bt. were screened at different temperatures and pHs for their hydrolytic potentials. By using a pair of specific primers, endochitinase gene was amplified from SBS Bt-5 strain through PCR, and then cloned into pTZ57 TA cloning vector and transferred in Escherichia coli DH5α strain. The sequenced gene (GenBank Accession No: HE995800) consists of 2031 nucleotides capable of encoding 676 residues. The protein consisted of three functional domains with a calculated molecular mass of 74.53 kDa and a pI value of 5.83. The amino acid sequence of chi gene showed 99% similarity to the genes of Bt MR11 endochitinase, Bt serovar kurstaki chitinase (kchi), Bt strain MR21 endochitinase and Bacillus cereus B4264.

Impact of Q139R substitution of MEB4-Cry2Aa toxin on its stability, accessibility and toxicity against Ephestia kuehniella

The Bacillus thuringiensis subsp. kurstaki strain MEB4 was previously found to be highly toxic to Ephestia kuehniella. SDS-PAGE analysis of the recombinant strain DH5α (pBS-cry2Aa-MEB4) showed that Cry2Aa-MEB4 delta-endotoxins were forming inclusion bodies, and were 2.75 fold more toxic towards E. kuehniella than those of Cry2Aa-BNS3. Besides to the 65kDa active toxin, proteolysis activation of Cry2Aa-BNS3 protein with E. kuehniella midgut juice generated an extra proteolysis form of 49kDa, which was the result of another chymotrypsin cleavage located in Leu144. The amino acid sequences alignment of Cry2Aa-MEB4 and Cry2Aa-BNS3 showed that among the different 15 amino acids, the Q139R substitution was found to be interesting. In fact, due to its presence within the loop α3-α4, the chymotrypsin-like protease was unable to access to its site in Cry2Aa-MEB4, resulting to the production of only the 65kDa form. The accessible surface and the stability studies of the structure model of the Cry2Aa-BNS3-49 form showed a lower hydrophobicity surface due to the omission of 144 amino acids from the N-terminal comparing with the active Cry2Aa-MEB4 protein. All these features caused the diminishing of Cry2Aa-BNS3 toxicity towards E. kuehniella.

Synergy Potential of Indole Alkaloids and Its Derivative against Drug-resistant Escherichia coli.

Antibacterial and synergy potential of naturally occurring indole alkaloids (IA): 10-methoxy tetrahydroalstonine (1), isoreserpiline (2), 10 and 11 demethoxyreserpiline (3), reserpiline (4), serpentine (5), ajmaline (6), ajmalicine (7), yohimbine (8), and α-yohimbine (9) was evaluated using microbroth dilution assay. Further, α-yohimbine (9) was chemically transformed into six semisynthetic derivatives (9A-9F), and their antibacterial and synergy potential in combination with nalidixic acid (NAL) against E.coli strains CA8000 and DH5α were also evaluated. The IA 1, 2, 4, 5, 9 and the derivative 9F showed eightfold reduction in the MIC of NAL against the DH5α and four- to eightfold reduction against CA8000. These alkaloids also reduced MIC of another antibiotic, tetracycline up to 8folds, against the MDREC-KG4, a multidrug-resistant clinical isolate of E.coli. Mode of action study of these alkaloids showed efflux pumps inhibitory potential, which was supported by their in silico binding affinity and downregulation of efflux pump genes. These results may be of great help in the development of cost-effective antibacterial combinations for treating patients infected with multidrug-resistant Gram-negative infections.

Expression of Recombinant pET22b-LysK-Cysteine/Histidine-Dependent Amidohydrolase/Peptidase Bacteriophage Therapeutic Protein in Escherichia coli BL21 (DE3)

ObjectivesBacteriophage-encoded endolysins are a group of enzymes that act by digesting the peptidoglycan of bacterial cell walls. LysK has been reported to lyse live staphylococcal cultures. LysK proteins containing only the cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) domain has the capability to show lytic activity against live clinical staphylococcal isolates, including methicillin-resistant Staphylococcus aureus (MRSA). The aim of this study was to clone and express LysK-CHAP domain in Escherichia coli BL21 (DE3) using pET22b as a secretion vector. The pET22b plasmid was used, which encoded a pelB secretion signal under the control of the strong bacteriophage T7 promoter. MethodsThe E. coli cloning strains DH5α and BL21 (DE3) were grown at 37°C with aeration in the Luria-Bertani medium. A plasmid encoding LysK-CHAP in a pET22b backbone was constructed. The pET22b vector containing LysK-CHAP sequences were digested with NcoI and HindIII restriction enzymes. Cloning accuracy was confirmed by electrophoresis. The pET22b-LysK plasmid was used to transform the E. coli strain BL21. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1mM to induce T7 RNA polymerase-based expression. Finally, western blot confirmed the expression of target protein. ResultsIn this study, after double digestion of pEX and pET22b vectors with HindIII and NcoI, LysK gene was cloned into two HindIII and NcoI sites in pET22b vector, and then transformed to E. coli DH5α. Cloning was confirmed with double digestion and analyzed with agarose gel. The recombinant pET22b-LysK plasmid was transformed to E. coli BL21 and the expression was induced by IPTG. The expression was confirmed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting method. Observation of a 28.5 kDa band confirmed LysK protein expression. ConclusionIn the present study, LysK-CHAP domain was successfully cloned and expressed at the pET22b vector and E. coli BL21 (DE3).