Multiple Cloning Site Research Articles

Elastin-like polypeptide-functionalized nanobody for column-free immunoaffinity purification of aflatoxin B1.

A column-free immunoaffinity purification (CFIP) technique for sample preparation of aflatoxin B1 (AFB1) was developed using an AFB1-specific nanobody (named G8) and an elastin-like polypeptide (ELP). The reversible phase transition between liquid and solid in response to temperature changes was exhibited by the ELP which was derived from human elastin. The G8 was tagged with ELPs of various lengths (20, 40, 60, and 80 repeat units) at the C-terminus using recursive directional ligation (RDL). Coding sequences were then subcloned into pET30a at the multiple cloning sites. Bioactive recombinant proteins were produced by expressing them as inclusion bodies in Escherichia coli BL21 (DE3), then dissolved and refolded. Analysis by indirect competitive enzyme-linked immunosorbent assay (icELISA) and transition temperature (Tt) measurement confirmed that the refolded G8-ELPs preserved the ability to recognize AFB1 as well as phase transition when the temperature rose above Tt. To establish the optimal conditions for cleaning AFB1, the effects of various parameters on recovery were investigated. The recovery in ELISA tests was 95 ± 3.67% under the optimized CFIP workflow. Furthermore, the CFIP-prepared samples were applied for high-performance liquid chromatography (HPLC) detection. The recovery in the CFIP-HPLC test ranged from 54 ± 1.86% to 98 ± 3.58% for maize, rice, soy sauce, and vegetable oil samples. To the best of our knowledge, this is the first report combining the function of both nanobody and ELP to develop a cleanup technique for small molecules in a complex matrix. The CFIP for the sample pretreatment was easy to use and inexpensive. In contrast to conventional immunosensitivity materials, the reagent utilized in the CFIP was entirely biosynthesized without any chemical coupling reactions. This suggests that the nanobody-ELP may serve as a useful dual-functional reagent for the development of sample cleaning or purification methods.

A nisin-inducible chromosomal gene expression system based on ICE Tn5253 of Streptococcus pneumoniae, transferable among streptococci and enterococci

The present work reports the development and validation of a chromosomal expression system in Streptococcus pneumoniae which permits gene expression under the control of Lactococcus lactis lantibiotic nisin. The system is based on the integrative and conjugative element (ICE) Tn5253 of S. pneumoniae capable of site-specific chromosomal integration and conjugal transfer to a variety of bacterial species. We constructed an insertion vector that integrates in Tn5251, an ICE contained in Tn5253, which carries the tetracycline resistance tet(M) gene. The vector contains the nisRK regulatory system operon, the L. lactis nisin inducible promoter PnisA upstream of a multiple cloning site for target DNA insertion, and is flanked by two DNA regions of Tn5251 which drive homologous recombination in ICE Tn5253. For system evaluation, the emm6.1::ha1 fusion gene was cloned and integrated into the chromosome of the Tn5253-carrying pneumococcal strain FR24 by transformation. This gene encodes a fusion protein containing the signal peptide, the 122 N-terminal and the 140 C-terminal aa of the Streptococcus pyogenes M6 surface protein joined to the HA1 subunit of the influenza virus A hemagglutinin. Quantitative RT-PCR analysis carried out on total RNA purified from nisin treated and untreated cultures showed an increase in emm6.1::ha1 transcript copy number with growing nisin concentration. The expression of M6-HA1 protein was detected by Western blot and quantified by Dot blot, while Flow cytometry analysis confirmed the presence on the pneumococcal surface. Recombinant ICE Tn5253::[nisRK]-[emm6.1::ha1] containing the nisin-inducible expression system was successfully transferred by conjugation in different streptococcal species including Streptococcus gordonii, S. pyogenes, Streptococcus agalactiae and Enterococcus faecalis. As for S. pneumoniae, the emm6.1::ha1 transcript copy number and the amount of M6-HA1 protein produced correlated with the nisin concentration used for induction in all investigated bacterial hosts. We demonstrated that this host-vector expression system is stably integrated as a single copy within the bacterial chromosome, is transferable to both transformable and non transformable bacterial species, and allows fine tuning of protein expression modulated by nisin concentration. These characteristics make our system suitable for a wide range of applications including complementation assays, physiological studies, host-pathogen interaction studies.

Expanding the genetic toolbox for the obligate human pathogen Streptococcus pyogenes.

Genetic tools form the basis for the study of molecular mechanisms. Despite many recent advances in the field of genetic engineering in bacteria, genetic toolsets remain scarce for non-model organisms, such as the obligatory human pathogen Streptococcus pyogenes. To overcome this limitation and enable the straightforward investigation of gene functions in S. pyogenes, we have developed a comprehensive genetic toolset. By adapting and combining different tools previously applied in other Gram-positive bacteria, we have created new replicative and integrative plasmids for gene expression and genetic manipulation, constitutive and inducible promoters as well as fluorescence reporters for S. pyogenes. The new replicative plasmids feature low- and high-copy replicons combined with different resistance cassettes and a standardized multiple cloning site for rapid cloning procedures. We designed site-specific integrative plasmids and verified their integration by nanopore sequencing. To minimize the effect of plasmid integration on bacterial physiology, we screened publicly available RNA-sequencing datasets for transcriptionally silent sites. We validated this approach by designing the integrative plasmid pSpy0K6 targeting the transcriptionally silent gene SPy_1078. Analysis of the activity of different constitutive promoters indicated a wide variety of strengths, with the lactococcal promoter P 23 showing the strongest activity and the synthetic promoter P xylS2 showing the weakest activity. Further, we assessed the functionality of three inducible regulatory elements including a zinc- and an IPTG-inducible promoter as well as an erythromycin-inducible riboswitch that showed low-to-no background expression and high inducibility. Additionally, we demonstrated the applicability of two codon-optimized fluorescent proteins, mNeongreen and mKate2, as reporters in S. pyogenes. We therefore adapted the chemically defined medium called RPMI4Spy that showed reduced autofluorescence and enabled efficient signal detection in plate reader assays and fluorescence microscopy. Finally, we developed a plasmid-based system for genome engineering in S. pyogenes featuring the counterselection marker pheS*, which enabled the scarless deletion of the sagB gene. This new toolbox simplifies previously laborious genetic manipulation procedures and lays the foundation for new methodologies to study gene functions in S. pyogenes, leading to a better understanding of its virulence mechanisms and physiology.

A low-cost and eco-friendly recombinant protein expression system using copper-containing industrial wastewater.

The development of innovative methods for highly efficient production of recombinant proteins remains a prominent focus of research in the biotechnology field, primarily due to the fact that current commercial protein expression systems rely on expensive chemical inducers, such as isopropyl β-D-thiogalactoside (IPTG). In our study, we designed a novel approach for protein expression by creating a plasmid that responds to copper. This specialized plasmid was engineered through the fusion of a copper-sensing element with an optimized multiple cloning site (MCS) sequence. This MCS sequence can be easily customized by inserting the coding sequences of target recombinant proteins. Once the plasmid was generated, it was introduced into an engineered Escherichia coli strain lacking copA and cueO. With this modified E. coli strain, we demonstrated that the presence of copper ions can efficiently trigger the induction of recombinant protein expression, resulting in the production of active proteins. Most importantly, this expression system can directly utilize copper-containing industrial wastewater as an inducer for protein expression while simultaneously removing copper from the wastewater. Thus, this study provides a low-cost and eco-friendly strategy for the large-scale recombinant protein production. To the best of our knowledge, this is the first report on the induction of recombinant proteins using industrial wastewater.

A Simple Allelic Exchange Method for Efficient Seamless Knockout of Up to 34-kbp-Long Gene Cassettes in Pseudomonas.

Gene knockout is a widely used technique for engineering bacterial genomes, investigating the roles of genes in metabolism, and conferring biological characteristics. Herein, we developed a rapid, efficient, and simple method for the knockout of long gene cassettes in Pseudomonas spp., based on a traditional allelic exchange strategy. The upstream and downstream sequences of the target gene cluster to be deleted were amplified using primers with 5'-end sequences identical to the multiple cloning sites of a suicide plasmid (mutant allele insert vector). The sequences were then fused with the linearized suicide plasmid in one step via seamless cloning. The resulting allelic exchange vector (recombinant plasmid) was introduced from the donor strain (Escherichia coli SM 10) into recipient cells (Pseudomonas putida, P. composti, and P. khazarica) via conjugation. Single-crossover merodiploids (integrates the vector into host chromosome by homologous recombination) were screened based on antibiotic resistance conferred by the plasmid, and double-crossover haploids (deleting the target gene clusters and inserted alien plasmid backbone) were selected using sucrose-mediated counterselection. Unlike other approaches, the method described herein introduces no selective marker genes into the genomes of the knockout mutants. Using our method, we successfully deleted polysaccharide-encoding gene clusters in P. putida, P. composti, and P. khazarica and generated four mutants with single-gene cassette deletions up to 18 kbp and one mutant with double-gene cassette deletion of approximately 34 kbp. Collectively, our results indicate that this method is ideal for the deletion of long genetic sequences, yielding seamless mutants of various Pseudomonas spp.

K fragment as a polymerase chain reaction-based vector for antibiotic resistance gene hunting

Antimicrobial resistance (AMR) has emerged as an escalating health issue in the global public health arena. To evaluate and predict AMR, it is of utmost importance to identify and characterize both the known and unknown genes responsible for AMR. While known genes can be readily detected, identifying unknown genes present a challenge. In this study, we developed the plasmid K fragment (pKF) by modifying the pUC19 vector, specifically by removing the multiple cloning site and introducing a Prom-RBS sequence. pKF was used for amplification of K fragment that contains a ribosomal binding site (RBS), and promoter at both ends and plasmid origin of replication. The functionality of added Prom-RBS sequence and K fragment as a cloning vector was tested by cloning chloramphenicol resistance gene amplicon and erythromycin resistance gene from genomic DNA, respectively. The cloning experiment demonstrated the usability of this newly developed cloning method with K fragment. K fragment is an innovative vector that can be easily obtained through amplification by polymerase chain reaction and lacks antibiotic resistance markers. This novel approach is convenient to use since it allows cloning of resistance genes at all orientations and this flexibility can be maneuvered by changing restriction enzymes for primers and fragments. With these distinctive features, this vector stands out to be a versatile tool for cloning both known and unknown resistance genes, and the improved method with K fragment enables the microbiological and molecular characterization of cloned genes. K fragment can be utilized for cloning of resistance genes in bacteria originated in different environments without having to perform bacterial culture. We believe that the convenience brought by this technique could lend itself efficient in the battle against the growing AMR crisis through pre-emptive identification of resistance genes.

Streamlined assembly of cloning and genome editing vectors for genus Clostridium

Reported herein is a new set of vectors designed to streamline molecular cloning and genome editing by exploiting modern cloning methods. The new vectors build on the existing pMTL8000 vectors that have been a staple of Clostridium research for more than a decade. The introduction of two pairs of type IIS restriction sites flanking an insulated multiple cloning site in both a cloning vector and a CRISPR-Cas9 gene editing vector enables plasmid construction in a "one-pot" reaction, avoiding the more laborious steps of conventional cloning. A synthetic lacZα expression cassette introduced between the cloning sites enables visual detection of background colonies. In addition, distinct selection markers on each vector permit selection of the desired clones according to antibiotic resistance. An example of strain development using the new vectors is demonstrated.

Increased lentivirus titer using ultra expression vectors

Lentivirus is an efficient gene transfer system that is widely used in basic science. We aimed to improve viral titer by applying an ultra-expression vectors to lentiviral packaging. Application of the ultra-expression vectors increased biological titer 4 times for standard preparation. We also evaluated the efficacy of the ultra-expression vectors to relatively longer insert fragments, such as CSII-CMV-CNROE containing 5 genes in multiple cloning sites. Packaging of the ultra-expression vectors showed 3.5 times higher biological titer compared with the original method. Our improved packaging system could be applied to lentivirus to produce higher titers.

Bacillus subtilis small replicative plasmid collection incorporating five distinct origins of replication and compatible with Golden Gate DNA assembly.

In order to facilitate Golden Gate DNA assembly, we have constructed a collection of Bacillus subtilis replicative plasmids representing five origins of replication derived from plasmids pUB110, pE194, pWV01, pBS72, and pTH1030. The first three of these plasmids use rolling circle replication and the latter two use theta replication. All of the plasmids carry the same multiple cloning site surrounded by transcriptional terminators. The plasmids are about three kilobases in size, allowing them to be easily amplified by inverse PCR using a common set of primers to generate cloning-ready amplicons. This plasmid PCR amplification approach also facilitates a workflow that eliminates Escherichia coli as a shuttle intermediate. All of the plasmids lack a site for at least three of the type IIS restriction enzymes BbsI, BsaI, Esp3I, PaqCI, or SapI, making them compatible with Golden Gate DNA assembly. We have demonstrated the utility of the plasmids by performing Golden Gate assembly of gusA and bgaB-reporter gene fragments and in expressing plasmid-borne red fluorescent protein under the control of RNA polymerase from bacteriophage K1E.

Development of an in vivo cleavable donor plasmid for targeted transgene integration by CRISPR-Cas9 and CRISPR-Cas12a

The CRISPR-Cas system is widely used for genome editing of cultured cells and organisms. The discovery of a new single RNA-guided endonuclease, CRISPR-Cas12a, in addition to the conventional CRISPR-Cas9 has broadened the number of editable target sites on the genome. Here, we developed an in vivo cleavable donor plasmid for precise targeted knock-in of external DNA by both Cas9 and Cas12a. This plasmid, named pCriMGET_9-12a (plasmid of synthetic CRISPR-coded RNA target sequence-equipped donor plasmid-mediated gene targeting via Cas9 and Cas12a), comprises the protospacer-adjacent motif sequences of Cas9 and Cas12a at the side of an off-target free synthetic CRISPR-coded RNA target sequence and a multiple cloning site for donor cassette insertion. pCriMGET_9-12a generates a linearized donor cassette in vivo by both CRISPR-Cas9 and CRISPR-Cas12a, which resulted in increased knock-in efficiency in culture cells. This method also achieved > 25% targeted knock-in of long external DNA (> 4 kb) in mice by both CRISPR-Cas9 and CRISPR-Cas12a. The pCriMGET_9-12a system expands the genomic target space for transgene knock-in and provides a versatile, low-cost, and high-performance CRISPR genome editing tool.

Vectors with a flexible multiple cloning site and modular epitope tags for gene expression studies in Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe is a widely used eukaryotic model organism for cell biology, genetics and metabolomics studies. S. pombe-specific autonomously replicating shuttle vectors are frequently used for gene expression studies because such vectors can stably replicate, and cells harbouring them can be easily selected. Vectors have been designed with varying promoter strengths and epitope tags, yet higher flexibility and diversity further augmenting their application is desired. Here, we have prepared a set of twenty vectors with auxotrophic markers for leucine and uracil by modifying the widely used pREP plasmid with a thiamine-repressible nmt1 (no message in thiamine) promoter. The vectors offer distinct benefits, including an improved multiple cloning site (MCS) with unique cutting sites for more restriction enzymes and a set of N- and C-terminal 3xMYC, 3xFLAG and EGFP epitope tags for visualization and purification of expressed proteins. Further, different constitutive and thiamine-repressible promoters are used for varying a gene's expression, as verified by western blot detection of vector-borne fusion proteins in S. pombe. Importantly, DNA sequences encoding various elements including the epitope tags are kept modular with respect to their flanking restriction sites. Vectors reported here enhance the repertoire of tools currently available for molecular studies in S. pombe.

Shikimate Kinase; Searching for Potential Novel Anti‐Tubercular Agents

Tuberculosis is a respiratory infection with over 10 million reported cases each year. This infection rate is responsible for over two million deaths annually, second only to HIV in fatalities among infectious diseases annually. Studies in the last decade have shown that tuberculosis rates have gradually increased. This observation suggests that there is an alarming increase in the prevalence of drug resistant strains of tuberculosis, thus the need for the discovery of novel antitubercular agents. The shikimate pathway is a seven step metabolic route that produces aromatic amino acids and other cellular metabolites. The target enzyme in this project, Mycobacterium tuberculosis Shikimate Kinase (MtSK), catalyzes the 5th step of this pathway. MtSK converts shikimate to shikimate 3 phosphate. The overall goal of this project is to express and characterize MtSK to screen for potential antitubercular agents. Initial methods included a bacterial transformation of XL1 blue competent E.coli cells. This preliminary transformation was performed using a pET‐21b plasmid with an aroK gene inserted into the multiple cloning site. Successfully transformed XL1 blue cells were cloned and a second transformation of BL21 DE3 competent cells was performed. Initial small scale expression showed the presence of a band around 20 kDa. The theoretical mass of the enzyme is 19.6 kDa which suggests MtSK was successfully expressed. Expression analysis for large scale supported data from small scale as a band of 20 kDa was present. The purity and molecular weight of MtSK were confirmed by nickel affinity chromatography as well as ESI‐LC‐MS. ATP‐dependent kinetic parameters determined via a spectrophotometric coupled assay showed a kcat of 50 ± 6 mM, KM of 0.2 ± 0.04 mM. shikimate dependent kinetic parameters showed a kcat of 55 ± 2 mM, KM of 1.8 ± 0.1 mM. Quenching intrinsic tyrosine fluorescence showed binding affinities similar to ATP and shikimate‐dependent parameters. The marine compound, avarone showed substantial quenching of intrinsic fluorescence. The inhibitory mechanism of avarone on MtSK is currently under investigation.

Development of an LB cloning system and its application in expression of fusion genes in Sphingomonas sp. WG

In order to overcome the challenges of insufficient restriction enzyme sites, and construct a fusion-expression vector with flexible fusion direction, we designed an LB cloning system based on the type IIS and type IIT restriction enzymes LguⅠ and BbvCⅠ. The LB cloning system is constructed by inserting the LB fragment (GCTCTTCCTCAGC) into the multiple cloning site region of the broad-host plasmid pBBR1MCS-3 using PCR. The LB fragment contains partially overlapped recognition sites of LguⅠ and BbvCⅠ. Therefore, the same non-palindromic sequence will be generated by these two restriction endonucleases digestion. This feature can be used to quickly and flexibly insert multiple genes into the expression vector in a stepwise and directed way. In order to verify the efficacy of the cloning system, two glycosyltransferase genes welB and welK of Sphingomonas sp. WG were consecutively fused to the LB cloning vector, and the recombinant plasmid was transferred into Sphingomonas sp. WG by triparental mating. The results showed that gene fusion expression has little effect on sphingan titer, but enhanced the viscosity of sphingan. The viscosity of the sphingan produced by recombinant strain Sphingomonas sp. WG/pBBR1MCS-3-LB-welKB was 24.7% higher than that of the wild strain after fermentation for 84 h, which would be beneficial for its application. In conclusion, the application of LB cloning system were verified using Sphingomonas sp. WG. The LB cloning system may provide an efficient tool for fusion expression of target genes.

Strawberry vein banding virus-based vector for transient overexpression in strawberry plants

Strawberry vein banding virus (SVBV) is a double-stranded DNA virus. In our previous studies, we generated an infectious clone of SVBV, pSVBV, which causes light-green vein banding symptoms along the leaf veins in strawberry plants (Fragaria vesca). In this study, we constructed pSVBV-P1-MCS and pSVBV-P4-MCS, two recombinant virus vectors containing a multiple cloning site (MCS) downstream of the SVBV-encoded movement protein gene (P1) and coat protein gene (P4), respectively. At 35 days post-inoculation, the two SVBV-based vectors could produce light-green vein banding symptoms on the systemic leaves of strawberry plants, indicating that they could successfully cause infection. Furthermore, the infectivity rates of the recombinant virus vectors pSVBV-P1-MCS and pSVBV-P4-MCS were similar to that of the wild-type infectious clone pSVBV, indicating that the insertion of MCS did not affect the infectivity of SVBV-based vectors. Additionally, we engineered SVBV as a transient overexpression vector, which could be used for the overexpression of exogenous green fluorescent protein in strawberry plants. Collectively, these SVBV-based vectors provide a new approach for the analysis of gene functions in strawberry plants.

Prediction of an Efficient Signal Peptide for Optimized Expression of Mycobacterium Tuberculosis Heparin-binding Hemagglutinin Gene in Periplasmic Compartment of Escherichia Coli: A Bioinformatics Investigation

Background: The heparin-binding hemagglutinin (HBHA) protein belonging to Mycobacterium tuberculosis is known as a molecular adjuvant. Objective: Hence, the expression of this protein in the prokaryotic system is essential. Methods: To predict an appropriate signal peptide for the expression of the HBHA protein, 50 signal peptides were selected from the signal peptide database. Then, the crucial parameters of signal peptides, including the probability of signal peptide, different regions of signal peptides, physicochemical features, sorting of signal peptides, and sub-cellular location were completely investigated by reliable tools. After the best-predicted signal peptide was identified, it was linked to the HBHA protein, and its secondary structure, tertiary structure, and in silico cloning in pET21a (+) was assessed. Findings: The results of different evaluations confirmed that only 13 signal peptides passed all features, including clearance of N, H, and C regions, D-score >0.7, instability index >40, and periplasmic localization. Finally, based on D-scores, among these 13 signal peptides, the asr (acid shock RNA) peptide with D-score=90 was selected as the best-predicted signal peptide to apply. Moreover, the results showed that the secondary structure of the adjuvant linked to asr peptide contained 88.18% alpha helix and 9.5% random coil. Also, the results of in silico cloning showed that the nucleotide sequences of the adjuvant linked to the asr peptide were successfully cloned between BamHI and XhoI enzymes in the multiple cloning site of pET21a (+). Conclusion: The results of this study confirmed that the asr peptide can be used as an appropriate signal peptide for the expression of the HBHA protein in the prokaryotic system.

Recombinant Rabies Virus With a Multiple Cloning Site: A Platform Capable of Immunological Alterations

Background: Rabies is fatal encephalitis, i.e., preventable by appropriate vaccination. Reverse genetics has proved promising for manipulating the rabies virus immunological characteristics. The insertion or deletion of a gene from the rabies genome could render specific functions to the rabies virus. Materials and Methods: A multiple cloning site including 111 nucleotides long harboring 10 single-cut restriction sites have been designed. The designed fragment was cloned between the G and L genes of the rabies virus genome. The recombinant rabies virus was rescued, and its infectivity was confirmed in the BHK-21 cell line. The recombinant virus propagation was compared with the initial rabies virus strain. Statistical analysis was performed using GraphPad Prism. Results: The cloning and localization of the multiple cloning site were verified by nucleotide sequencing. The recombinant virus properly propagated and rescued in the BHK-21 cell line. Comparing the recombinant virus with the initial rabies virus has shown that both viruses had similar functionality and propagation rate. Conclusion: The recombinant virus obtained in the present study could facilitate further cloning experiments. Examples include constructing a marker virus, carrying green fluorescent protein to be used either in rabies immunity assays or tracking the virus infection in relevant tissues.

A Myo-Inositol-Inducible Expression System for Corynebacterium glutamicum and Its Application.

Corynebacterium glutamicum is one of the important industrial microorganisms for production of amino acids and other value-added compounds. Most expression vectors used in C. glutamicum are based on inducible promoter (P tac or P trc ) activated by isopropyl-β-D-thiogalactopyranoside (IPTG). However, these vectors seem unsuitable for large-scale industrial production due to the high cost and toxicity of IPTG. Myo-inositol is an ideal inducer because of its non-toxicity and lower price. In this study, a myo-inositol-inducible expression vector pMI-4, derived from the expression vector pXMJ19, was constructed. Besides the original chloramphenicol resistance gene cat, multiple cloning sites, and rrnB terminator, the pMI-4 (6,643 bp) contains the iolR q cassette and the myo-inositol-inducible promoter P iolT1 . The pMI-4 could stably replicate in the C. glutamicum host. Meanwhile, the non-myo-inositol degradation host strain C. glutamicumΔiolGΔoxiCΔoxiDΔoxiE for maintaining the pMI-4 was developed. Overexpression of hemA M and hemL using pMI-4 resulted in a significant accumulation of 5-aminolevulinic acid, indicating its potential application in metabolic engineering and industrial fermentation.

Production of SARS-CoV-2 Virus-Like Particles in Insect Cells.

Coronavirus disease (COVID-19) causes a serious threat to human health. Virus-like particles (VLPs) constitute a promising platform in SARS-CoV-2 vaccine development. In this study, the E, M, and S genes were cloned into multiple cloning sites of a new triple expression plasmid with one p10 promoter, two pPH promoters, and three multiple cloning sites. The plasmid was transformed into DH10 BacTM Escherichia coli competent cells to obtain recombinant bacmid. Then the recombinant bacmid was transfected in ExpiSf9TM insect cells to generate recombinant baculovirus. After ExpiSf9TM cells infection with the recombinant baculovirus, the E, M, and S proteins were expressed in insect cells. Finally, SARS-CoV-2 VLPs were self-assembled in insect cells after infection. The morphology and the size of SARS-CoV-2 VLPs are similar to the native virions.

Tuberculosis shikimate kinase as a target for the development of new antimycobacterials

Tuberculosis is a respiratory infection with over 10 million reported cases each year. This infection rate is responsible for over two million deaths annually, second only to HIV in fatalities among infectious diseases. Since 1960 tuberculosis rates have steadily declined worldwide as medical technology has advanced to produce more effective antibiotics. However, recent studies have shown that tuberculosis rates have seen a steady increase. This observation suggests that there is an alarming increase in the prevalence of drug-resistant strains of tuberculosis, thus the need for the discovery of novel anti-tubercular agents. Targeting potential enzymatic pathways for drug discovery requires the pathway to possess minimal overlap with the host. The shikimate pathway is a seven-step metabolic route that produces aromatic amino acids and other cellular metabolites. This pathway is typically present in microorganisms and has no mammalian counterpart, making any of the enzymes in this pathway suitable targets for screening of potential anti-tubercular agents. The target enzyme here, Mycobacterium tuberculosis Shikimate Kinase (MtSK), catalyzes the 5th step of this pathway, converting shikimate to shikimate-3-phosphate. The overall goal of this project is to express and characterize MtSK in order to screen for potential anti-tubercular agents. Initial methods included a bacterial transformation of XL-1 blue competent E.coli cells. This preliminary transformation was performed using a pET-21b plasmid with an aroK gene inserted at the multiple cloning site. Successfully transformed XL-1 blue cells were cloned and a second transformation using BL21 DE3 competent cells as an expression host was performed. Small scale expression showed the presence of a band around 20 kDa. The theoretical mass of the enzyme is 19.6 kDa which suggests MtSK was successfully expressed within the transformed cells. Expression analysis for large-scale supported data from small scale as a band of 20 kDa once again appeared in the verification SDS-PAGE gel. Purification of MtSK was carried out through nickel affinity chromatography. Subsequently, kinetic characterization and inhibitors studies will be performed using inhibitors like avarone and hymenidin.

Independent DNA analysis in biochemistry labs expands student perceptions of plasmid architecture

In order to assess independent skills in DNA analysis, undergraduate students enrolled in three biochemistry lab courses isolated distinct plasmids from E. coli host bacteria using silica-coated iron magnetic nanoparticles and determined purity using spectrometry. The various plasmids originated from the AddGene Fire vector kit, such that no two students isolated the same plasmid DNA. After assessing nucleic acid purity, students independently selected restriction enzymes and anticipated fragment lengths of DNA that emerged with electrophoresis on an agarose gel. This initial experience with DNA analysis was further supported with queries of plasmid architecture features. Since every student analyzed a distinct plasmid, independent effort was necessary with appropriate peer collaboration. The lab experience, interfaced with the AddGene database, demanded 100% student engagement, while exposing students to an expanded representative set of plasmids that included segments coding for antibiotic resistance, green fluorescent protein, lacZ, and multiple cloning sites. In all cases, students responded favorably to the experience.