Plasmid Clones Research Articles

Identification of a novel alternate promoter element in the pheST operon of Escherichia coli.

Earlier work in this laboratory revealed that fitA was same as pheS as a recombinant clone, pSRJ5R1 harboring pheS+ gene complemented transcription-defective fitA76 Ts (temperature sensitive) mutant. However, this clone lacked the native promoter (NP)of pheST operon. A putative - 10 promoter like element was suggested to act as promoter in this clone. This work investigated the veracity of this putative promoter as well as its downstream regulatory region towards driving the pheS expression. Plasmid clones with promoter-mutations or -deletions were constructed by PCR-based cloning and their ability to complement fitA76 Ts mutant strains was checked. Chromosomal mutations were transferred into various genetic backgrounds via P1-transductions. Relative viability assays were performed to check the extent of complementation. Clones harboring point mutations (PM-pheS) or deletion (PD1-pheS) of - 10 region of the putative promoter did not abolish complementation of the fitA76 Ts phenotype. Subsequently, a novel alternate promoter (AP)was discovered by downstream deletion clone (PD2-pheS) which failed to complement. Keeping PD1-pheS intact but mutating initiation codon of pheS (ATG→TTG) failed to complement. Complementation ability of novel alternate promoter is poor in HfrC strain background unlike native promoter which complements well independent of strain background. A novel alternate-promoter of pheST operon was identified by mutational/deletional analyses and earlier reported putative - 10 promoter was shown to be dispensable. Alternate promoter is relA dependent.

Viral coexistence and insertional mutations in the ORF8 region of SARS-CoV-2: A possible mechanism of nucleotide insertion

The virus obtained from a swab sample ID: S66 in Hiroshima was reported to have a single T-base insertion in the ORF8 coding region. However, no T insertion was observed when we determined the genomic sequence using another method. We then extracted RNA from the S66 swab sample and sequenced the insertion site using the Sanger method. The resulting waveform was disrupted beyond the insertion site, suggesting the presence of a mixed population of viruses with different sequences. Through plasmid cloning of RT-PCR amplification fragments and virus cloning by limiting dilution, along with TIDE analysis to determine the ratio of components from the Sanger sequencing waveform, it was confirmed that the sample contained a mixture of viruses with varying numbers of T-base insertions. The virus with one T insertion (T1+) was predominant in 70–75 % of the genomes, and genomes with T0, T2+, T3+, T4+, and T5+ were also detected. No T-base insertion mutations were observed in the ORF8 region in three other SARS-CoV-2 samples. In the S66 sample, a C27911T point mutation near the insertion site in the ORF8 region resulted in a sequence of seven or more consecutive T bases, which was the cause of the T-base insertion. When the cloned S66 virus (T1+) was passaged in cultured cells, there was a tendency for viruses with more insertion bases to become dominant with successive generations, suggesting that the T-base insertion was due to polymerase stuttering. The insertion of T bases resulted in synthesis of deletion mutants of the ORF8 protein, but no significant change was observed in the proliferation of the viruses in cultured cells. A search of the GenBank database using NCBI BLAST for viruses similar to S66 with T-base insertion mutations revealed hundreds of viruses widely distributed on the molecular phylogenetic tree. These base insertion viruses were thought to have occasionally arisen during the virus infection process. This study suggests one mechanism of insertion mutations in SARS-CoV-2, and it is important to consider the emergence of future mutant strains.

PSLBII-12 Cloning and analysis of target-specific CRISPR RNA and screening primers to create β-lactoglobulin knock-out goat fibroblast cell lines for nuclear transfer

Abstract Goat milk and meat are primary or additional source of income for small farmers in large parts of the world. Goat farming is increasing in southeastern United States, due to influx of ethnic populations. Goat milk is rich in health promoting nutrients compared with other livestock milk; however, its consumption results in allergenicity in humans due to presence of β-lactoglobulin protein in milk. Although some efforts have been made to eliminate allergenicity by modifying β-lactoglobulin gene, especially in Chinese goats, more research is needed to make it a reality. The objective of this study was to design, clone and characterize CRISPR RNA (crRNA) in a plasmid vector containing Cas 9 expression cassette as well as design and analyze PCR primers for efficient screening of edited genomes as a prelude to create and analyze β-lactoglobulin knock-out goats. Three crRNAs, two from exon 1 and one from exon 2 of goat β-lactoglobulin gene with 5 base hangovers were designed and cloned individually in GeneArt CRISPR Nuclease plasmid Vector. Sequence analysis of selected plasmid clones using U 6 promoter primer, upstream of the crRNA insert, confirmed that at least 3 clones of each crRNA had the right sequence. Additionally, 4 sets (forward and reverse) of primers to amplify 4 loci in β-lactoglobulin gene flanking the crRNA sequences in goat genome were designed. PCR amplification using genomic DNA isolated from goat blood as well as goat skin fibroblasts (GSF) confirmed expected single bands of 655, 812, 502 and 437 bps with respective primer sets. Transfection of 3 crRNA clones in GSF cells and their analysis for site-specific genome modification is in progress. In conclusion, we have designed, cloned, and tested crRNA in a plasmid vector along with 4 sets of PCR primers to screen for edited genomes which will be useful to create β-lactoglobulin edited goats to eliminate milk allergenicity.

Multi-amplicon nitrogen cycling gene standard: An innovative approach for quantifying N-transforming soil microbes in terrestrial ecosystems

Agriculture accounts for nearly three-fourths of all nitrous oxide (N2O) anthropogenic emissions, a potent greenhouse gas (GHG). Improvements in agricultural management practices are critical for improving plant nitrogen (N) use efficiency and reducing N2O emissions. While implementing “climate-smart” management can reduce N2O emissions, building a linkage between these emissions and the N-cycling microbes that drive them will require N-cycling gene assays that are cost-effective, practical, and accessible to climate-smart researchers. In a recent study, a group of researchers demonstrated the suitability of synthetic oligonucleotides to serve as real-time quantitative PCR (qPCR) standards for quantifying microbial genes in soils. While this study is not based on that study, our results complement their findings and offer fresh insights into this novel methodology. Here, we describe an innovative approach that provides precision in the absolute quantification of five N-transforming target genes (AOB amoA, AOA amoA, nirK, nirS, and nosZ) in soils. Similar to the aforementioned study, our method obviates the cost-intensive aspects of traditional qPCR methods for preparing standard curves in soil microbial ecology (e.g., gel purification of PCR products and in-vivo cloning of plasmids) and is capable of being extended to any biochemical system, or environment of interest. Additionally, our qPCR approach provides several advantages: it utilizes a single multi-amplicon synthetic oligonucleotide, requires only a single dilution series and one stock solution to quantify all gene targets, and contains a non-edaphic gene (ZIKV) that can serve as a soil spike to determine DNA extraction efficiency and DNA recovery estimates. Furthermore, our protocol mitigates PCR inhibition from soil-derived substances using a modified DNA extraction protocol and inhibitor-tolerant polymerase. Ultimately, our work aims to reduce the technical burden on non-biologists/biochemists when conducting soil microbiological assays, support the development of climate mediation practices, and advance climate-smart agriculture goals.

Expression and characterization of a novel β-1,4-endoglucanase from Bacillus subtilis strain isolated from a pulp and paper mill wastewater

The production of fermentable sugars from lignocellulosic biomass is achieved by the synergistic action of a group of enzymes called cellulases. Cellulose is a long chain of chemically linked glucoses by β-1,4 bonds. The enzyme β-1,4-endoglucanase is the first cellulase involved in the degradation, breaking the bond of the amorphous regions. A β-1,4-endoglucanase enzyme with high activity was obtained from a Bacillus subtilis strain isolated from wastewater of a pulp and paper mill. Sequencing and bioinformatic analysis showed that the gene amplified by PCR consisting of 1407 nucleotides and coding for a β-1,4-endoglucanase enzyme of approximately 55 kDa. The open reading frame (ORF) encoding the mature endoglucanase (eglS) was successfully inserted in a modified cloning plasmid (pITD03) and into the pYD1 plasmid used for its expression in yeast. Carboxymethylcellulose (CMC) plate assay, SDS-PAGE, and zymogram confirmed the production and secretion by the transformed E. coli BL21-SI strain of a 39 kDa β-1,4-endoglucanase consistent with the catalytic domain without the cellulose-binding module (CBM). The results showed that the truncated β-1,4-endoglucanase had higher activity and stability.

Rapid Rescue of Goose Astrovirus Genome via Red/ET Assembly.

The host-specific infection of Avian Astrovirus (AAstVs) has posed significant challenges to the poultry industry, resulting in substantial economic losses. However, few reports exist on the functional consequences of genome diversity, cross-species infectivity and mechanisms governing virus replication of AAstVs, making it difficult to develop measures to control astrovirus transmission. Reverse genetics technique can be used to study the function of viruses at the molecular level, as well as investigating pathogenic mechanisms and guide vaccine development and disease treatment. Herein, the reverse genetics technique of goose astrovirus GAstV/JS2019 strain was developed based on use of a reconstructed vector including CMV promotor, hammerhead ribozyme (HamRz), hepatitis delta virus ribozyme (HdvRz), and SV40 tail, then the cloned viral genome fragments were connected using Red/ET recombineering. The recombinant rGAstV-JS2019 was readily rescued by transfected the infectious clone plasmid into LMH cells. Importantly, the rescued rGAstV/JS2019 exhibited similar growth kinetics comparable to those of the parental GAstV/JS2019 isolate in cultured cells. Our research results provide an alternative and more effective reverse genetic tool for a detailed understanding of viral replication, pathogenic mechanisms, and molecular mechanisms of evolution.

Long noncoding RNA GPRC5D-AS1 in renal cell carcinoma: a molecular mechanism study.

Clear cell renal cell carcinoma (RCC) is the most common subtype of RCC. Although targeted therapy can provide superior treatment outcomes, it is prone to drug resistance, and individual responses to immunotherapy vary greatly. Therefore, finding new diagnostic and therapeutic targets for RCC is of considerable importance. Long noncoding RNA (lncRNA) GPRC5D-AS1 can serve as a biomarker in clinical applications and the prognosis of lung squamous cell carcinoma. However, the specific mechanism of action of lncRNA GPRC5D-AS1 in RCC has not yet been clarified. Therefore, this paper explores the expression of lncRNA GPRC5D-AS1 in the renal cancer cell line 786-0, and conducts a preliminary study of its molecular mechanism. Selecting nude mice for tumor experiments is because of the high genomic and physiological similarity between mice and humans. Conducting tumor research on mice allows for better control of experimental conditions, aiding researchers in more accurately observing and analysing tumor characteristics and responses. Small interfering RNA (siRNA) and plasmid cloning DNA (pcDNA) 3.1 were used to transfect renal cancer cell line 786-0 to silence and overexpress the lncRNA GPRC5D-AS1 gene. Quantitative real-time fluorescence polymerase chain reaction was used to detect the difference in lncRNA GPRC5D-AS1 expression in blank control group, negative control group, siGPRC5D-AS1 group and oeGPRC5D-AS1 group. The effects of silence and overexpression of lncRNA GPRC5D-AS11 on the proliferation of 786-0 cells were detected in cell colony formation experiments; the changes in the migration and invasion of 786-0 cells were detected via cell scratch assay and transwell assay, respectively; the differences in tumor growth between groups were determined via tumorigenesis experiments in nude mice; and the expression of proliferation-related protein [β-catenin, Ki67 and proliferating cell nuclear antigen (PCNA)] and invasion-related protein (N-cadherin and E-cadherin) were detected via Western blotting. Compared with blank control group and negative control group, the siGPRC5D-AS1 group showed a significant decrease in the relative expression of lncRNA GPRC5D-AS1 (P<0.05), a significant increase in the number of proliferating cells and migrating cells (P<0.05), a significant increase in the tumor volume of nude mice (P<0.05), a significant increase in β-catenin, Ki67, PCNA and N-cadherin protein expression (P<0.05), and a significant decrease in E-cadherin protein expression (P<0.05); conversely, these results were opposite for the eGPRC5D-AS1 group. Silencing the expression of lncRNA GPRC5D-AS1 can enhance the proliferation, invasion, and migration ability of renal cancer cell line 786-0, which can be weakened by the overexpression of lncRNA GPRC5D-AS1.

Engineering probiotic bacteria to express tcdB antigen as an oral vaccine carrier against Clostridium difficile infection

Background Clostridium (now known as Clostridioides) difficile (C. difficile) is a spore-forming, gram-positive organism that can pose serious public health complications. The elderly are especially vulnerable to C. difficile infections, which can be fatal. C. difficile strains cause symptomatic diseases via the release of two toxins; tcdA and tcdB, that induce inflammation and tissue damage. Vaccines targeting any of these toxins may offer an effective strategy against C. difficile infections. Objective This study aimed to use live probiotics as oral vaccines to express the C. difficile toxin B gene. Oral vaccination has many advantages over intramuscular injection, as it has higher compliance, feasibility, and simpler administration. In addition, oral vaccines can stimulate both mucosal and systemic immunity against the target antigen. Probiotic bacteria were chosen as they present ideal candidates for this goal in terms of safety and health promotion. Materials and methods We chose two probiotic strains: Lactobacillus gasseri ATCC 33323 in addition to Enterococcus faecium NM1015, which had previously been identified in our lab and is capable of suppressing C. difficile colonization. The C-terminal of the tcdB gene was amplified by polymerase chain reaction (PCR) from C. difficile, cloned, and transformed into an E. coli EZ strain. The tcdB fragment was digested with BamHI and XhoI (NEB, UK) and subcloned into the bile salt-inducible expression plasmid pLB210 (obtained from INRA, France). The transformation and electroporation procedures were employed to insert cloning and expression plasmids into the target bacteria. Colony PCR was used to confirm the engineering strains. Reverse transcription PCR was used to confirm the expression of the C. difficile tcdB fragment. Results and conclusion The expression vector ‘p210-tcdB’ was constructed, then introduced into the selected probiotic strains by electroporation and confirmed by colony PCR and plasmid extraction. The reverse transcription (RT)-PCR confirmed the expression of the gene by the engineered strains. No significant difference in the survival rate was observed between the engineered strains and their parental types at pH 2.00 and 1% oxygen. Moreover, the strains showed satisfactory plasmid stability for 210 generations. Future work will involve the in vivo evaluation of the engineered probiotic strains as oral vaccines against C. difficile using an animal model.

Silver-Coordinated Watson-Crick Pairing-Driven Three-Dimensional DNA Walker for Locus-Specific Detection of Genomic N6-Methyladenine and N4-Methylcytosine at the Single-Molecule Level.

N6-Methyladenine (6mdA) and N4-methylcytosine (4mdC) are the two most dominant DNA modifications in both prokaryotes and eukaryotes, but standard hybridization-based techniques cannot be applied for the 6mdA/4mdC assay. Herein, we demonstrate the silver-coordinated Watson-Crick pairing-driven three-dimensional (3D) DNA walker for locus-specific detection of genomic 6mdA/4mdC at the single-molecule level. 6mdA-DNA and 4mdC-DNA can selectively hybridize with the binding probes (BP1 and BP2) to form 6mdA-DNA-BP1 and 4mdC-DNA-BP2 duplexes. The 6mdA-C/4mdC-A mismatches cannot be stabilized by AgI, and thus, 18-nt BP1/BP2 cannot be extended by the catalysis of KF exonuclease. Through toehold-mediated strand displacement (TMSD), the signal probe (SP1/SP2) functionalized on the gold nanoparticles (AuNPs) can competitively bind to BP1/BP2 in 6mdA-DNA-BP1/4mdC-DNA-BP2 duplex to obtain SP1-18-nt BP1 and SP2-18-nt BP2 duplexes. The resulting DNA duplexes can act as the substrates of lambda exonuclease, leading to the cleavage of SP1/SP2 and the release of Cy3/Cy5 and 18-nt BP1/BP2. The released 18-nt BP1/BP2 can subsequently serve as the walker DNA, moving along the surface of the AuNP to activate dynamic 3D DNA walking and releasing abundant Cy3/Cy5. The released Cy3/Cy5 can be quantified by single-molecule imaging. This nanosensor exhibits high sensitivity with a limit of detection (LOD) of 9.80 × 10-15 M for 6mdA-DNA and 9.97 × 10-15 M for 4mdC-DNA. It can discriminate 6mdA-/4mdC-DNA from unmodified genomic DNAs, distinguish 0.01% 6mdA-/4mdC-DNA from excess unmethylated DNAs, and quantify 6mdA-/4mdC-DNA at specific sites in genomic DNAs of liver cancer cells and Escherichia coli plasmid cloning vector, providing a new platform for locus-specific analysis of 6mdA/4mdC in genomic DNAs.

Irreversible light-activated SpyLigation mediates split-protein assembly in 4D.

The conditional assembly of split-protein pairs to modulate biological activity is commonly achieved by fusing split-protein fragments to dimerizing components that bring inactive pairs into close proximity in response to an exogenous trigger. However, current methods lack full spatial and temporal control over reconstitution, require sustained activation and lack specificity. Here light-activated SpyLigation (LASL), based on the photoregulation of the covalent SpyTag (ST)/SpyCatcher (SC) peptide-protein reaction, assembles nonfunctional split fragment pairs rapidly and irreversibly in solution, in engineered biomaterials and intracellularly. LASL introduces an ortho-nitrobenzyl(oNB)-caged lysine into SC's reactive site to generate a photoactivatable SC (pSC). Split-protein pairs of interest fused to pSC and ST are conditionally assembled via near-ultraviolet or pulsed near-infrared irradiation, as the uncaged SC can react with ST to ligate appended fragments. We describe procedures for the efficient synthesis of the photocaged amino acid that is incorporated within pSC (<5 days) as well as the design and cloning of LASL plasmids (1-4 days) for recombinant protein expression in either Escherichia coli (5-6 days) or mammalian cells (4-6 days), which require some prior expertise in protein engineering. We provide a chemoenzymatic scheme for appending bioorthogonal reactive handles onto E. coli-purified pSC protein (<4 days) that permits LASL component incorporation and patterned protein activation within many common biomaterial platforms. Given that LASL is irreversible, the photolithographic patterning procedures are fast and do not require sustained light exposure. Overall, LASL can be used to interrogate and modulate cell signaling in various settings.

A Directed Evolution Protocol for Engineering Minimal Transcription Factors, Based on CIS Display.

Directed evolution is an efficient strategy for obtaining desired biomolecules. Since the 1990s, the emergence of display techniques has enabled high-throughput screening of functional proteins. However, classical methods require library construction by plasmid cloning and are limited by transformation efficiencies, typically limiting library sizes to ~106-107 variants. More recently, in vitro techniques have emerged that avoid cloning, allowing library sizes of >1012 members. One of these, CIS display, is a DNA-based display technique which allows high-throughput selection of biomolecules in vitro. CIS display creates the genotype-phenotype link required for selection by a DNA replication initiator protein, RepA, that binds exclusively to the template from which it has been expressed. This method has been successfully used to evolve new protein-protein interactions but has not been used before to select DNA-binding proteins, which are major components in mammalian synthetic biology. In this chapter, we describe a directed evolution method using CIS display to efficiently select functional DNA-binding proteins from pools of nonbinding proteins. The method is illustrated by enriching the minimal transcription factor Cro from a low starting frequency (1 in 109). This protocol is also applicable to engineering other DNA-binding proteins or transcription factors from combinatorial libraries.

Development of highly discriminatory SCoT- and CBDP-based SCAR fingerprint for authentication of Indian senna (Senna alexandrina Mill.) formerly Cassia angustifolia Vahl.).

Indian senna (Senna alexandrina Mill.) (formerly Cassia angustifolia Vahl.) is an important medicinal plant of the family Fabaceae. The leaves and pods of Indian senna yield sennosides and rhein-based laxative. Adulteration of Indian senna is a serious issue as with most of the medicinal plants used in the Indian systems of traditional medicine. The bulk of dried leaves and pods of morphologically related species, such as Cassia fistula, Senna occidentalis, Senna sophera, and Senna tora, is usually mixed with those of the Indian senna, and the admixture is used in laxative-based formulations. The present investigation is a modest attempt at developing species-specific start codon targeted (SCoT) polymorphism- and CAAT-box-derived polymorphism (CBDP)-based sequence-characterized amplified region (SCAR) markers for the identification and authentication of Indian senna and four adulterant species (C. fistula, S. occidentalis, S. sophera, and S. tora species). In this study, genomic DNA extracted from 44 accessions of Indian senna and four adulterant species was subjected to SCoT and CBDP PCR. The polymorphic amplicons were identified, eluted, ligated, and transformed into Escherichia coli DH5 α strain. PCR, restriction analysis, and DNA sequencing confirmed the transformed recombinant plasmid clones. Post-sequencing, the sequence of the primary SCoT and CBDP primers was analyzed and extended into the unique signature sequence of the concerned accessions. This resulted in development of one SCoT-44- and two CBDP-25-based SCARs. SCoT-44 SCAR produced a signature amplicon of 287 bp for accession DCA120, and CBDP-25 SCAR yielded signature amplicons of 575 and 345 bp for accessions DCA13 and DCA119, respectively. The developed SCAR markers were validated across 48 samples (44 accessions of Indian senna and 4 adulterant species) and produced distinct amplicons in Indian senna only, while no such amplicon was observed in the other four adulterant species. The information generated using these markers have been faithfully converted to single-locus, unequivocal, highly reproducible, and informative sequence-based SCAR markers. These markers will enable discrimination of individual plants on the basis of unique sequence-specific amplicons, which could be used as diagnostic markers to settle issues pertaining to the true identity of Indian senna.

Induction of proteome changes involved in the cloning of mcr-1 and mcr-2 genes in Escherichia coli DH5-α strain to evaluate colistin resistance

Plasmid genes, termed mobile colistin resistance-1 (mcr-1) and mobile colistin resistance-2 (mcr-2), are associated with resistance to colistin in Escherichia coli (E. coli). These mcr genes result in a range of protein modifications contributing to colistin resistance. This study aims to discern the proteomic characteristics of E. coli-carrying mcr-1 and mcr-2 genes. Furthermore, it evaluates the expression levels of various proteins under different conditions (with and without colistin). Plasmid extraction was performed using an alkaline lysis-based plasmid extraction kit, whereas polymerase chain reaction was used to detect the presence of mcr-1 and mcr-2 plasmids. The E. coli DH5α strain served as the competent cell for accepting and transforming mcr-1 and mcr-2 plasmids. We assessed proteomic alterations in the E. coli DH5α strain both with and without colistin in the growth medium. Proteomic data were analysed using mass spectrometry. The findings revealed significant protein changes in the E. coli DH5α strain following cloning of mcr-1 and mcr-2 plasmids. Of the 20 proteins in the DH5α strain, expression in 8 was suppressed following transformation. In the presence of colistin in the culture medium, 39 new proteins were expressed following transformation with mcr-1 and mcr-2 plasmids. The proteins with altered expression play various roles. The results of this study highlight numerous protein alterations in E. coli resulting from mcr-1 and mcr-2-mediated resistance to colistin. This understanding can shed light on the resistance mechanism. Additionally, the proteomic variations observed in the presence and absence of colistin might indicate potential adverse effects of indiscriminate antibiotic exposure on treatment efficacy and heightened pathogenicity of microorganisms.

Overexpression of microRNA-205-5p promotes cholangiocarcinoma growth by reducing expression of homeodomain-interacting protein kinase 3

The microRNA miR-205-5p has diverse effects in different malignancies, including cholangiocarcinoma (CCA), but its effects on CCA progression is unclear. Here we investigated the role and function of miR-205-5p in CCA. Three CCA cell lines and human serum samples were found to have much higher expression levels of miR-205-5p than seen in typical cholangiocyte cell lines and healthy controls. Inhibition of miR-205-5p suppressed CCA cell motility, invasion and proliferation of KKU-213B whereby overexpression of miR-205-5p promoted cell proliferation and motility of KKU-100 cells. Bioinformatics tools (miRDB, TargetScan, miRWalk, and GEPIA) all predicted various miR-205-5p targets. Experiments using miR-205-5p inhibitor and mimic indicated that homeodomain-interacting protein kinase 3 (HIPK3) was a potential direct target of miR-205-5p. Overexpression of HIPK3 using HIPK3 plasmid cloning DNA suppressed migration and proliferation of KKU-100 cells. Notably, HIPK3 expression was lower in human CCA tissues than in normal adjacent tissues. High HIPK3 expression was significantly associated with longer survival time of CCA patients. Multivariate regression analysis indicated tissue HIPK3 levels as an independent prognostic factor for CCA patients. These findings indicate that overexpression of miR-205-5p promotes CCA cells proliferation and migration partly via HIPK3-dependent way. Therefore, targeting miR-205-5p may be a potential treatment approach for CCA.

The roles of 6K protein on Getah virus replication and pathogenicity.

Alphavirus is a type of arbovirus that can infect both humans and animals. The amino acid sequence of the 6K protein, being one of the structural proteins of the alphavirus, is not conserved. Deletion of this protein will result in varying effects on different alphaviruses. Our study focuses on the function of the Getah virus (GETV) 6K protein in infected cells and mice. We successfully constructed infectious clone plasmids and created resulting viruses (rGETV and rGETV-Δ6K). Our comprehensive microscopic analysis revealed that the 6 K protein mainly stays in the endoplasmic reticulum. In addition, rGETV-Δ6K has lower thermal stability and sensitivity to temperature than GETV. Although the deletion of the 6K protein does not reduce virion production in ST cells, it affects the release of virions from host cells by inhibiting the process of E2 protein transportation to the plasma membrane. Subsequent in vivo testing demonstrated that neonatal mice infected with rGETV-Δ6K had a lower virus content, less significant pathological changes in tissue slices, and milder disease than those infected with the wild-type virus. Our results indicate that the 6K protein effectively reduces the viral titer by influencing the release of viral particles. Furthermore, the 6K protein play a role in the clinical manifestation of GETV disease.

642. The impact of novel E1:M407I mutation on replicative fitness of the Chikungunya virus ECSA-IOL strain during infection of mammalian and mosquito cells,

Abstract Background Chikungunya virus (CHIKV) is an Alpha virus which is transmitted by Aedes genus of mosquitoes. It causes febrile illness with severe arthralgia, myalgia and rashes. Since 2000, the re-emergence of this pathogen have made it a global health security threat as it has caused severe form of diseases with epidemics of large magnitude and also spread to European and American continent. Studies have identified different point mutations in E1 and E2 region associated with virulence and infectivity, which is behind these severe epidemics. Pakistan reported first CHIKV outbreak with more than 30,000 infections from its largest city in 2016 which is already endemic for Dengue virus (DENV). Methods 32 CHIKV PCR-positive serum samples were collected from molecular pathology section of DUHS followed by RNA extraction and amplifcation of E1 region by PCR. The amplified products were prurified and sanger sequenced by BigDye termination method. Mutation analyses &amp; Bayesian phylogeny was done by bioinformatic tools. CHIKV E1:M407I mutation was cloned in to CHIKV wild type infectious cDNA clone plasmid (SL-CK-1) by site directed mutagenesis (SDS). In vitro viral RNA was synthesized after plasmid linearization, which was electroporated into VERO-CCL-81 cells. M407I mutant virus was recovered from the culture, titrated by plaque assay and sanger sequenced to confirmed the M407I mutation. Viral competition assay was performed in mammalian and mosquitoes cell lines to determine the relative replicative fitness of mutant CHIKV virus over wild type. Results Sequence analysis revealed 7 unique mutations in E1 including one mutation (M407I) in more than half of the sequenced samples. Phylogenetic analyses revealed this outbreak was caused by CHIKV-IOL genotype with possible inception from India. Virus competition assay showed relative replicative fitness for CHIKV E1:M407I mutant in Vero-CLL-81 and AAG2 cells have greater advantage while in C6/36, it has slight advantage over wild type virus. Conclusion This study reports a novel mutations in E1 protein of CHIKV with fitness advantage over wild type strain. Phylogeny revealed the outbreak was caused by A226-IOL strain with possible origin from ancestral strain of Indian outbreak. Disclosures All Authors: No reported disclosures

Optimizing the Metal Bioreduction Process in Recombinant Shewanella azerbaijanica Bacteria: A Novel Approach via mtrC Gene Cloning and Nitrate-Reducing Pathway Destruction.

Environmental pollution is growing every day in terms of the increase in population, industrialization, and urbanization. Shewanella azerbaijanica is introduced as a highly potent bacterium in metal bioremediation. The mtrC gene was selected as a cloning target to improve electron flux chains in the EET (extracellular electron transfer) pathway. Using the SDM (site-directed mutagenesis) technique, the unique gene assembly featured the mtrC gene sandwiched between two napD/B genes to disrupt the nitrate reduction pathway, which serves as the primary metal reduction competitor. Shew-mtrC gene construction was transferred to expression plasmid pET28a (+) in the expression host bacteria (E. coli BL21 and S. azerbaijanica), in pUC57, cloning plasmid, which was transferred to the cloning host bacteria E. coli Top10 and S. azerbaijanica. All cloning procedures (i.e., synthesis, insertion, transformation, cloning, and protein expression) were verified and confirmed by precise tests. ATR-FTIR analysis, CD, western blotting, affinity chromatography, SDS-PAGE, and other techniques were used to confirm the expression and structure of the MtrC protein. The genome sequence and primers were designed according to the submitted Shewanella oneidensisMR-1 genome, the most similar bacteria to this native species. The performance of recombinant S. azerbaijanica bacterium in metal bioremediation, as sustainable strategy, has to be verified by more research.

Construction and functional verification of size-reduced plasmids based on TMP resistance gene dfrB10.

Plasmid size is one of the factors affecting transfection efficacy in most of the molecular genetic research studies. One effective approach for reducing plasmid size is to replace relatively large, conventional antibiotic resistance genes with the short-size dfrB10 gene. The successful construct of a series of dfrB10-based tool plasmids and their functional validation, via comparison with original plasmids, suggest that dfrB10 is a potent drug resistance selection marker. The antibiotic trimethoprim offers convenient usage comparable to that of ampicillin or kanamycin. Additionally, fluorescence analysis has demonstrated the compatibility of TMP with protein expression in various host cells. Based on these findings, TMP-dfrB10 could be an alternative choice for future use in molecular genetic research studies that require miniature plasmids to achieve optimal results.

Molecular characterization of highly prevalent Escherichia coli and Escherichia marmotae resistant to extended-spectrum cephalosporins in European starlings (Sturnus vulgaris) in Tunisia.

European starlings are widespread migratory birds that have already been described as carrying bacteria resistant to extended-spectrum cephalosporins (ESC-R). These birds are well known in Tunisia because they spend the wintertime in this country and are hunted for human consumption. The goal of our study was to estimate the proportion of ESC-R in these birds and to characterize the collected isolates using whole-genome sequencing. Results showed that 21.5% (42/200) of the birds carried either an extended-spectrum beta-lactamase (ESBL) or an acquired AmpC gene. Diverse bla CTX-M genes were responsible for the ESBL phenotype, bla CTX-M-14 being the most prevalent, while only bla CMY-2 and one bla CMY-62 were found in AmpC-positive isolates. Likewise, different genetic determinants carried these resistance genes, including IncHI2, and IncF plasmids for bla CTX-M genes and IncI1 plasmids for bla CMY-2 genes. Three chromosomally encoded bla CTX-M-15 genes were also identified. Surprisingly, species identification revealed a large proportion (32.7%) of Escherichia marmotae isolates. This species is phenotypically indistinguishable from Escherichia coli and has obviously the same capacity to acquire ESC-R genes. Our data also strongly suggest that at least the IncHI2/pST3 plasmid can spread equally between E. coli and E. marmotae. Given the potential transmission routes between humans and animals, either by direct contact with dejections or through meat preparation, it is important to closely monitor antimicrobial resistance in European starlings in Tunisia and to set up further studies to identify the sources of contamination of these birds. IMPORTANCE The One Health concept highlighted knowledge gaps in the understanding of the transmission routes of resistant bacteria. A major interest was shown in wild migratory birds since they might spread resistant bacteria over long distances. Our study brings further evidence that wild birds, even though they are not directly submitted to antibiotic treatments, can be heavily contaminated by resistant bacteria. Our results identified numerous combinations of resistance genes, genetic supports, and bacterial clones that can spread vertically or horizontally and maintain a high level of resistance in the bird population. Some of these determinants are widespread in humans or animals (IncHI2/pST3 plasmids and pandemic clones), while some others are less frequent (atypical IncI1 plasmid and minor clones). Consequently, it is essential to be aware of the risks of transmission and to take all necessary measures to prevent the proportions of resistant isolates from increasing uncontrollably.

A human papillomavirus whole genome plasmid repository: A resource for HPV DNA quality control reagents.

Well characterized reference reagents are useful for assay validation, proficiency/competency assessment, daily run controls, and to improve inter-laboratory comparisons. Synthetic human papillomavirus (HPV) DNA fragments and plasmid clones are available, but synthetic fragments include limited segments of the HPV genome and many HPV plasmids have interrupted coding regions or contain partial genomes. As a result, they are not compatible with all HPV DNA detection and typing assays. To address this need, we are establishing an HPV plasmid repository of HPV clones containing the whole genome of each type with no interruptions in coding regions. To date, HPV plasmid clones for 16 HPV types, (including all vaccine types and 14 types in clinical assays: HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) have been constructed using a Gibson assembly method and validated by sequencing and the Novaplex HPV typing assay. The newly constructed HPV whole genome plasmids can serve as a quality control reagent resource for HPV DNA assays and are available for public health and research laboratories.