Methods for modification of target nucleic acids. The method involves a construct in which guide RNA is covalently linked to donor RNA (fusion NA) to be introduced into the target nucleic acid by homologous recombination and is based on the introduction of a nuclease, e.g. CRISPR or TALEN, into the cell containing the target nucleic acid. The fusion NA may be introduced as a DNA vector.

An alkaline protease mutant, and a gene, engineered strain, a preparation method and application thereof are provided. The method comprises the following steps of extracting genome DNA of Bacillus clausii, performing PCR amplification to obtain a wild-type alkaline protease gene sequence, mutating the wild-type alkaline protease gene obtained by the amplification through an error-prone PCR, performing high-throughput screening to obtain a plurality of highly active alkaline protease genes, performing DNA shuffling on the highly active alkaline protease genes, and performing screening to obtain eight alkaline protease mutant genes with higher activity.

The present invention provides a pyruvate-responsive biosensor and a construction method and use thereof. In the present invention, pyruvate-responsive biosensors with different dynamic ranges are successfully constructed by optimizing the PdhR binding sequence inserted on the P43 promoter and optimizing the insertion site, wherein the minimum increase in dynamic range is by 0.6 time, and the maximum increase is by 30.7 times. The pyruvate-responsive biosensors are useful in the precise control of the expression of each gene in the cell. Since pyruvate is a key metabolite of central carbon in the cells, these biosensors are capable of dynamically regulating the expression level of intracellular genes according to changes in the content of pyruvate in the cells, thereby achieving the dynamic control of intracellular metabolic flux. The pyruvate biosensor obtained in the present invention has a good specificity, and a response range to pyruvate of 10-35 nmol/g DCW.

The present invention provides a pyruvate-responsive biosensor and a construction method and use thereof. In the present invention, pyruvate-responsive biosensors with different dynamic ranges are successfully constructed by optimizing the PdhR binding sequence inserted on the P43 promoter and optimizing the insertion site, wherein the minimum increase in dynamic range is by 0.6 time, and the maximum increase is by 30.7 times. The pyruvate-responsive biosensors are useful in the precise control of the expression of each gene in the cell. Since pyruvate is a key metabolite of central carbon in the cells, these biosensors are capable of dynamically regulating the expression level of intracellular genes according to changes in the content of pyruvate in the cells, thereby achieving the dynamic control of intracellular metabolic flux. The pyruvate biosensor obtained in the present invention has a good specificity, and a response range to pyruvate of 10-35 nmol/g DCW.

Disclosed herein are methods for the production of low to medium expressing constitutive promoters in bacteria and promoters produced therewith.

Disclosed herein are methods for the production of low to medium expressing constitutive promoters in bacteria and promoters produced therewith.

The invention relates to, in part, improved methods for the production of beta-lactamase using Escherichia coli (E. coli) cells. High yield production of beta-lactamase is achieved using methods of the invention.

The invention relates to, in part, improved methods for the production of beta-lactamase using Escherichia coli (E. coli) cells. High yield production of beta-lactamase is achieved using methods of the invention.

Methods of expressing an expression product of interest are provided. Accordingly there is provided a method comprising introducing into a cell a polynucleotide comprising an AimR responsive element operatively linked to a nucleic acid sequence encoding the expression product of interest, and contacting said cell with an AimP peptide comprising an amino acid sequence of XXXXGG/A, wherein said AimP peptide is capable of binding said AimR polypeptide and dissociating said AimR polypeptide from said AimR responsive element. Also provided are articles of manufacture, isolated peptides, polynucleotides and nucleic acid constructs.

Methods of expressing an expression product of interest are provided. Accordingly there is provided a method comprising introducing into a cell a polynucleotide comprising an AimR responsive element operatively linked to a nucleic acid sequence encoding the expression product of interest, and contacting said cell with an AimP peptide comprising an amino acid sequence of XXXXGG/A, wherein said AimP peptide is capable of binding said AimR polypeptide and dissociating said AimR polypeptide from said AimR responsive element. Also provided are articles of manufacture, isolated peptides, polynucleotides and nucleic acid constructs.