Disclosed herein are compositions and methods for modifying a bacterial population. In some embodiments, described herein is a bacteriophage comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, provided that the bacteriophage is rendered lytic.

Methods and systems for autoinduction of gene expression, without the need to add exogenous inducers. A dual genetic element system, which includes a first, high copy number genetic element comprising a first gene of interest that is under the control of an inducible promoter, and a second, low copy number genetic element comprising a gene encoding a transcriptional factor which, upon expression, regulates transcription from the inducible promoter, wherein activation of transcription from the inducible promoter does not require addition of an exogenous inducer.

Methods and systems for autoinduction of gene expression, without the need to add exogenous inducers. A dual genetic element system, which includes a first, high copy number genetic element comprising a first gene of interest that is under the control of an inducible promoter, and a second, low copy number genetic element comprising a gene encoding a transcriptional factor which, upon expression, regulates transcription from the inducible promoter, wherein activation of transcription from the inducible promoter does not require addition of an exogenous inducer.

A protein expression system for use in a prokaryotic host is provided, the expression system comprising: a) an expression cassette comprising a nucleic acid sequence encoding a protein of interest operably linked to a T7 RNA polymerase-dependent promoter; and b) an expression cassette comprising a nucleic acid sequence encoding T7 RNA polymerase operably linked to a host polymerase-dependent λ phage promoter and a single perfect palindrome operator sequence; wherein the expression cassette for T7 RNA polymerase is located on the chromosome of a host cell.

A protein expression system for use in a prokaryotic host is provided, the expression system comprising: a) an expression cassette comprising a nucleic acid sequence encoding a protein of interest operably linked to a T7 RNA polymerase-dependent promoter; and b) an expression cassette comprising a nucleic acid sequence encoding T7 RNA polymerase operably linked to a host polymerase-dependent λ phage promoter and a single perfect palindrome operator sequence; wherein the expression cassette for T7 RNA polymerase is located on the chromosome of a host cell.

The present disclosure relates generally to bacterial delivery vehicles for use in efficient transfer of a desired payload into a target bacterial cell. More specifically, the present disclosure relates to bacterial delivery vehicles with desired host ranges based on the presence of a chimeric receptor binding protein (RBP) composed of a fusion between the N-terminal region of a RBP derived from a lambda-like bacteriophage and the C-terminal region of a different RBP.

The present disclosure relates generally to bacterial delivery vehicles for use in efficient transfer of a desired payload into a target bacterial cell. More specifically, the present disclosure relates to bacterial delivery vehicles with desired host ranges based on the presence of a chimeric receptor binding protein (RBP) composed of a fusion between the N-terminal region of a RBP derived from a lambda-like bacteriophage and the C-terminal region of a different RBP.

Some aspects of this disclosure provide methods for phage-assisted continuous evolution (PACE) of proteases. Some aspects of this invention provide methods for evaluating and selecting protease inhibitors based on the likelihood of the emergence of resistant proteases as determined by the protease PACE methods provided herein. Some aspects of this disclosure provide strategies, methods, and reagents for protease PACE, including fusion proteins for translating a desired protease activity into a selective advantage for phage particles encoding a protease exhibiting such an activity and improved mutagenesis-promoting expression constructs. Evolved proteases that recognize target cleavage sites which differ from their canonical cleavage site are also provided herein.

A Cas9 expression plasmid, a genome editing system and a genome editing method for Escherichia coli are provided. The Cas9 expression plasmid includes a tracrRNA sequence, a Cas9 gene and a chloramphenicol resistance gene (Cm.sup.R). The Cas9 expression plasmid is applied to CRISPR/Cas-coulped λ-red recombineering system for editing genomes of E. coli with high efficiency.

A Cas9 expression plasmid, a genome editing system and a genome editing method for Escherichia coli are provided. The Cas9 expression plasmid includes a tracrRNA sequence, a Cas9 gene and a chloramphenicol resistance gene (Cm.sup.R). The Cas9 expression plasmid is applied to CRISPR/Cas-coulped λ-red recombineering system for editing genomes of E. coli with high efficiency.