dnaI

Abstract: This invention provides a cell comprising a DNA molecule having a regulatory element from a gene, other than a gene encoding a green fluorescent protein operatively linked to a DNA sequence encoding the green fluorescent protein. This invention also provides living organisms which comprise the above-described cell. This invention also provides a method for selecting cells expressing a protein of interest which comprises: a) introducing into the cells a DNAI molecule having DNA sequence encoding the protein of interest and DNAII molecule having DNA sequence encoding a green fluorescent protein; b) culturing the introduced cells under conditions permitting expression of the green fluorescent protein and the protein of interest; and c) selecting the cultured cells which express green fluorescent protein, thereby selecting cells expressing the protein of interest. Finally, this invention provides various uses of a green fluorescent protein.

Abstract: Over evolutionary time bacteriophages have developed unique proteins that arrest critical cellular processes to commit bacterial host metabolism to phage reproduction. Here, we apply this concept of phage-mediated bacterial growth inhibition to antibiotic discovery. We sequenced 26 Staphylococcus aureus phages and identified 31 novel polypeptide families that inhibited growth upon expression in S. aureus. The cellular targets for some of these polypeptides were identified and several were shown to be essential components of the host DNA replication and transcription machineries. The interaction between a prototypic pair, ORF104 of phage 77 and DnaI, the putative helicase loader of S. aureus, was then used to screen for small molecule inhibitors. Several compounds were subsequently found to inhibit both bacterial growth and DNA synthesis. Our results suggest that mimicking the growth-inhibitory effect of phage polypeptides by a chemical compound, coupled with the plethora of phages on earth, will yield new antibiotics to combat infectious diseases.

Abstract: Fifty temperature-sensitive mutants defective in DNA synthesis at high temperature have been identified among 655 temperature-sensitive mutants isolated at random from a mutagenised population of B. subtilis. They are distributed in a non-random fashion in 9 genetic linkage groups, located in different regions of the B. subtilis genome. It is suggested that at least 14 genes are involved in B. subtilis DNA replication.

Abstract: Once an engineered organism completes its task, it is useful to degrade the associated DNA to reduce environmental release and protect intellectual property. Here we present a genetically encoded device (DNAi) that responds to a transcriptional input and degrades user-defined DNA. This enables engineered regions to be obscured when the cell enters a new environment. DNAi is based on type-IE CRISPR biochemistry and a synthetic CRISPR array defines the DNA target(s). When the input is on, plasmid DNA is degraded 10(8)-fold. When the genome is targeted, this causes cell death, reducing viable cells by a factor of 10(8). Further, the CRISPR nuclease can direct degradation to specific genomic regions (for example, engineered or inserted DNA), which could be used to complicate recovery and sequencing efforts. DNAi can be stably carried in an engineered organism, with no impact on cell growth, plasmid stability or DNAi inducibility even after passaging for >2 months.