Technologies that permit controlled changes in the expression of genes are immensely important for biological research and for biotechnological applications. Type IE Cascade/CRISPR system based kit is developed to achieve transcriptional gene silencing. This kit offers all-in-one vector for gene silencing in different bacterial strains and species. The vector provided in the kit is capable of expressing both the Cascade and crRNA array required for modulating gene expression. The GFP reporter control plasmid and pCascr_GFP plasmid which expresses crRNA against GFP reporter plasmid is also provided in the kit. This serves as positive control assay for gene silencing experiment in the chosen host system. The specificity of Cascade binding is provided by the integral crRNA component, which can be easily designed to target virtually any stretch of DNA.

1. Programmable, easy to use and efficient gene silencing tool.
2. Stand-alone silencing platform capable of functioning in an individual cell.
3. Titrable and reversible gene silencing.
4. Silencing of two genes simultaneously possible.
5. Active in different bacterial species and strains.
6. Silencing of target locus on plasmid/or chromosome individually or simultaneously.
7. Can be used for understanding function of essential genes, metabolic flux tuning of enzymes involved in different pathways or to study biological function of hypothetical genes.

Technologies that permit controlled changes in the expression of genes are immensely important for biological research and for biotechnological applications. Type IE Cascade/CRISPR system based comprehensive kit is developed to achieve transcriptional gene silencing. This kit offers all-in-one vector for gene silencing in different bacterial strains and species. The vector provided in the kit is capable of expressing both the Cascade and crRNA array required for modulating gene expression. The GFP reporter control plasmid and pCascr_GFP plasmid which expresses crRNA against GFP reporter plasmid is also provided in the kit. This serves as positive control assay for gene silencing experiment in the chosen host system. The specificity of Cascade binding is provided by the integral crRNA component, which can be easily designed to target virtually any stretch of DNA.

1. Programmable, easy to use and efficient gene silencing tool.
2. Stand-alone silencing platform capable of functioning in an individual cell.
3. Titrable and reversible gene silencing.
4. Silencing of two genes simultaneously possible.
5. Active in different bacterial species and strains.
6. Silencing of target locus on plasmid/or chromosome individually or simultaneously.

1. Understanding the function of essential genes.
2. Metabolic flux tuning by modulating expression of enzymes involved in different pathways.
3. Study biological function of hypothetical ORFs/ putative annotations.
4. Confirm in-vivo function of previously assigned gene functions by other in-vitro techniques.
5. Understand interplay of two different genes in one particular function.

The use of this kit is not limited to applications described here. This can be used for answering or expanding range of basic and applied questions and can be adapted to additional organisms and cell systems.

1. The subunits of Cascade from E. coli K-12 strain are expressed as operon under an inducible promoter.
2. The expression of synthetic crRNA array is driven by strong constitutive J23119-speI promoter.
3. The crRNA array has two spacer sequences enfolded between repeat sequences.
4. BsaI and BsmBI restriction enzyme sites for cloning of spacers with in the crRNA array.
5. The vector allows seamless redefining of new target location via. one step short oligo based cloning of spacer sequence.
6. Ampicillin resistance marker for selection in different bacterial strains.

Microbiology lab for growing of E. coli culture and extraction of plasmid.

Space

1. Working space: 40 sq. meter

Raw Material

1. Easily available within the country

Manpower

1. One technician.