The present disclosure relates to the field of genetic engineering, especially relates to a xylose-induced genetically engineered bacteria used for producing ectoine as well as a construction method and use thereof The genetically engineered bacteria is constructed by heterologously expressing the ectABC gene cluster from Halomonas elongata on the E. coli chromosome, using the promoter of xylose transporter coding gene xylF to control the RNA polymerase from T7 bacteriophage, reconstructing a synthesis pathway of ectoine and constructing a plasmid-free system, and enhancing the expression of target genes by a strong promoter T7; the yiled of ectoine reached 12-16 g/L after 20-28 h fermentation in shake flask, and reached 35-50 g/L after 24-40 h fermentation in a 5 L fermentor.

The present invention embraces an RNA replicon (self-amplifying RNA vector (saRNA)) that can be replicated by a replicase of a self-replicating virus, e.g., a replicase of alphavirus origin. According to the invention, translation of the replicase open reading frame is uncoupled from a 5′-terminal cap by placing translation of the replicase open reading frame under the translational control of an internal ribosome entry site (IRES). Thereby the initiation of translation depends on the molecular properties of the respective IRES, which compared to cap-dependent translation may require less or no cellular initiation factors to direct the ribosome to the translational start site. According to the invention, IRES-controlled replicase translation may allow the use of uncapped synthetic saRNA. Furthermore, the use of an IRES provides for the option to insert additional transgenes upstream to the IRES.

Disclosed herein is a pH activated nanoparticle that can be used to deliver labile therapeutic or diagnostic agents to the cytoplasm of cells. These nanoparticles allow the agents to escape the endosome by releasing a gas in an amount effective to disrupt the endosome and release the agents into the cytoplasm. The disclosed nanoparticles have a shell, such as a phospholipid bilayer shell, and a core containing a gas bound to a substrate by a pH sensitive interaction. Also disclosed herein is are methods for delivering a pH sensitive cargo to the cytoplasm of a cell, treating triple negative breast cancer (TNBC) in a subject, and treating HER2+ breast cancer in a subject.

This invention generally relates to a novel composition of RNA/mRNA medicines as well as vaccines produced by using replicase- and/or RNA-dependent RNA polymerase (RdRp)-mediated RNA cycling reaction (RCR). The present invention is useful for developing a variety of self-amplifying RNA/mRNA (samRNA) medicines and vaccines containing at least a replicase/RdRp-binding site in the 5′- or 3′-end, or both, of any desired RNA molecule, including but not limited to antisense RNA (aRNA), small interferring RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA)/miRNA precursor, long non-coding RNA (lnRNA) and mRNA. These RNA molecules can be either in single-stranded or in double-stranded, or mixed, conformation. The samRNA so obtained is useful not only for producing RNA-based vaccines and/or medicines but also for generating the mRNA-associated proteins, peptides, and/or antibodies under a proper in-vitro or in-cell translation condition. The replicase/RdRp-binding sites used in samRNA are derived or modified from coronaviral (e.g. COVID-19) and/or hepatitis C viral (HCV) RNA-dependent RNA polymerases (RdRp) in either single-stranded or double-stranded compositions.

The present disclosure relates to the field of genetic engineering, especially relates to a xylose-induced genetically engineered bacteria used for producing ectoine as well as a construction method and use thereof. The genetically engineered bacteria is constructed by heterologously expressing the ectABC gene cluster from Halomonas elongata on the E. coli chromosome, using the promoter of xylose transporter coding gene xylF to control the RNA polymerase from T7 bacteriophage, reconstructing a synthesis pathway of ectoine and constructing a plasmid-free system, and enhancing the expression of target genes by a strong promoter T7; the yield of ectoine reached 12-16 g/L after 20-28 h fermentation in shake flask, and reached 35-50 g/L after 24-40 h fermentation in a 5 L fermentor.

Provided is a method for replicating a mirror nucleic acid, comprising: reacting a mirror nucleic acid template, a mirror nucleic acid primer and mirror dNTPs/rNTPs in the presence of a mirror nucleic acid polymerase, so as to obtain the mirror nucleic acid.

The present invention relates to a genetic determinant which may comprise at least two copies of a combination of two closely linked RDR1 genes, which two closely linked RDR1 genes are inversely oriented, and which genetic determinant leads to virus resistance when present in a plant. In one embodiment, of the RDR1 genes in the combination is represented by SEQ ID NO: 1 or has at least 70% sequence identity, and one of the RDR1 genes in the combination is represented by SEQ ID NO: 3 or has at least 70% sequence identity; or one of the RDR1 genes in the combination encodes a protein represented by SEQ ID NO: 2 or a protein that has at least 70% sequence identity, and one of the RDR1 genes encodes a protein represented by SEQ ID NO: 4 or a protein that has at least 70% sequence identity.

Another aim of the current invention may include a recombinant cell-free expression system, the reaction mixture containing all the cell-free reaction components necessary for the in vitro biosynthesis of biological compounds, proteins, enzymes, biosimilars or chemical modification of small molecules.

The present invention enables simultaneous and stable expression of a plurality of foreign genes by using a stealthy RNA gene expression system that is a complex that does not activate the innate immune mechanism and is formed from an RNA-dependent RNA polymerase, a single-strand RNA binding protein, and negative-sense single-strand RNAs including the following (1) to (8): (1) a target RNA sequence that codes for any protein or functional RNA; (2) an RNA sequence forming a noncoding region and derived from mRNA expressed in animal cells; (3) a transcription initiation signal sequence recognized by the RNA-dependent RNA polymerase; (4) a transcription termination signal sequence recognized by the polymerase; (5) an RNA sequence containing a replication origin recognized by the polymerase; (6) an RNA sequence that codes for the polymerase and of which codons are optimized for the species from which an introduction target cell is derived; (7) an RNA sequence that codes for a protein for regulating the activity of the polymerase and of which codons are optimized for the species from which the introduction target cell is derived; and (8) an RNA sequence that codes for the single-strand RNA binding protein and of which codons are optimized for the species from which the introduction target cell is derived.

This disclosure provides, among other things, amplifiable nucleic acid constructs for expressing a gene of interest in a cell, e.g., an erythroid cell. The amplifiable nucleic acid construct may contain the gene of interest and an RNA-dependent RNA polymerase (RdRP)-responsive 5′ UTR, and may optionally further contain an RdRP-responsive 3′ UTR. RdRP may also be provided, e.g., on the same construct or a different construct.