The disclosure belongs to the field of biotechnology, and in particular to a blocking ELISA kit for detecting an antibody to swine acute diarrhea syndrome coronavirus (SADS-CoV) N protein. The kit includes an enzyme plate coated with SADS-CoV N protein, an HRP-labeled mouse anti-SADS-CoV N protein monoclonal antibody (mAb), a positive serum control, and a negative serum control. The kit may detect positive sera diluted at 1:512, with no cross-reaction with positive sera against porcine epidemic diarrhea virus (PEDV), transmissible gasteroenteritis virus (TGEV) and porcine deltacoronavinis (PDCoV) etc., and the intrabatch and interbatch coefficient of variation is less than 10%. The comparison result with the indirect immunofluorescence test shows that the concordance rate of the blocking ELISA kit of the present disclosure is 99.6%, the Kappa value is 0.91, and the blocking ELISA method established by the present disclosure is highly consistent with IFA.

Provided herein are methods for the production of a vector with a size of at least 16 kb from bacterial cells comprising the consecutive steps of a) obtaining bacterial cells comprising a vector with a size of at least 16 kb, comprising an inducible origin of replication, b) inoculating culture medium with the bacterial cells comprising the vector, c) culturing the bacterial cells in the culture medium, d) adding one or more inducers of said inducible origin of replication to the culture medium when the bacterial culture has reached an optical density at 600 nm (OD600) of at least 20, e) further culturing the bacterial cells in the culture medium, f) optionally separating the bacterial cells from the culture medium, and g) recovering the plasmid from the bacterial cells. Also provided herein are vectors with a size of at least 16 kb comprising an inducible origin of replication for use in such methods.

The technology relates to a method for producing a transformed and fully-segregated cyanobacteria, the method comprising incubating the cyanobacteria and a nucleic acid comprising a selectable marker under conditions suitable for transformation of the cyanobacteria with the nucleic acid; further incubating the cyanobacteria in growth media under conditions suitable for recovery of the cyanobacteria; and selecting the transformed and fully-segregated cyanobacteria using a selection agent.

It is disclosed herein that (a) an anti-tumor DNA vaccine delivered using a MIP DNA vector is a less effective tumor treatment than the corresponding anti-tumor DNA vaccine delivered using a conventional pDNA vector, despite the MIP DNA vector eliciting a higher frequency of antigen-specific CD8+ T cells; and (b) tumor infiltrating CD8+ T cells in animals immunized with the MIP DNA vector express higher levels of the immune checkpoint protein LAG-3 than animals immunized with a conventional pDNA vector, while the expression levels of other immune checkpoint proteins was the same for both groups. Based on these findings, improved methods and compositions for administering DNA vaccines are disclosed. Specifically, DNA vaccines delivered with MIP DNA are administered along with a LAG-3 pathway blocking agent, resulting in a more effective vaccine-induced cellular immune response.

The invention provides for systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are vectors and vector systems, some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for selecting specific cells by introducing precise mutations utilizing the CRISPR/Cas system.

Embodiments of the present invention provide compositions and methods for recombinantly generating tagless constructs of proteins or peptides. In certain embodiments, recombinant proteins or peptides disclosed herein concern human papilloma virus (HPV). Other embodiments concern using these constructs in compositions to elicit immune responses in a subject to one or more HPV types. Therapeutic and prophylactic vaccines for the prevention and treatment of viral infections are also disclosed. Nucleic acids and expression vectors coding for constructs contemplated herein are provided. In certain embodiments, an HPV capsid protein generated is devoid of any fusion tags. In addition, truncated forms of HPV L1 are contemplated.

The present invention relates to a covalently closed circular recombinant DNA molecule comprising an origin of replication and an insert comprising a homopolymeric region, wherein the homopolymeric region is located at a distance of least 500 bp from the origin of replication in the direction of replication and/or wherein the insert comprising a homopolymeric region is oriented so that the direction of transcription of the insert is the same as the direction of replication of the origin of replication. The invention further relates to the use of the covalently closed circular recombinant DNA molecule for increasing the yield and/or shortening the fermentation time during fermentation.

Disclosed are nucleic acid sequences comprising a first E. coli homology region, wherein the first E. coli homology region comprises a protospacer adjacent motif (PAM) mutation; a constitutive promoter; a mevalonate-3-kinase (M3K) gene; a mevalonate diphosphate decarboxylase (MVD) gene; and a second E. coli homology region. Disclosed are vectors comprising one or more of the disclosed nucleic acid sequences. Disclosed are recombinant cells comprising a nucleic acid sequence, wherein the nucleic acid sequence comprises a first E. coli homology region, wherein the first E. coli homology region comprises a PAM mutation; a constitutive promoter; a M3K gene; a MVD gene; and a second E. coli homology region.

An autoinducer-2 (AI-2) molecular response-based starting element and an Escherichia coli (E. coli) dynamic regulation system and method constructed thereby are provided. A cell density-dependent starting element P.sub.J23119-LsrR-P.sub.lsrA based on an AI-2 molecular response is constructed. The element can be used to self-induce the expression of dCpf1, and crRNAs of different target genes are further assembled, such that the self-inducible element can be used for dCpf1-CRP to achieve the dynamic regulation of genes in a synthesis pathway. In the present disclosure, vectors pACYDuet-P.sub.J23119-LsrR-P.sub.lsrA-dCpf1-CRP, pRSFDuet-GFP-mCherry, and pETDuet-crRNA can be constructed to simultaneously achieve the transcriptional activation and inhibition of different genes. The construction method of recombinant E. coli in the present disclosure is simple and has promising application prospects.

A method for modulating a target organism in a microbiome, comprising contacting the microbiome with a cis-conjugative plasmid that can replicate and conjugate with organisms in the microbiome including the target organism, the conjugative plasmid comprising conjugation genes and a gene or a combination of genes capable of being expressed in the target organism and that only modulates the target organism in the microbiome. Also the isolated cis-conjugative plasmid comprising conjugation genes and a gene or a combination of genes capable of being expressed in a target bacteria within a microbiome or biofilm and that modulates the target bacteria in the microbiome or biofilm.