"
Team:HKU-Hong Kong/Project
From 2011.igem.org
| Super Silencer |
| Silencing of genes is a method adopted by cells to control their gene expression and the production of gene products. In eukaryotic cells, both heterochromatin and euchromatin are present. There is active transcription in euchromatin because of acetylation while there is inactive transcription in heterochromatin
because of methylation. Our project is called “Super Silencer”. We are trying to mimic heterochromatin in Escherichia coli (E. coli) so that we can better manipulate the bacteria by silencing particular genes. Our idea is to utilize a natural protein called histone-like nucleoid structuring protein (H-NS) to silence the target gene. H-NS is a major bacterial chromatin component which influences DNA structure and gene expression. It binds to DNA non-specifically with its C-terminal and polymerizes by its N-terminal. We are trying to replace the H-NS's C-ternimal with a specific DNA binding domain like tetR to perform a site specific DNA binding to the corresponding DNA binding site tetO. We anticipate that the mutated H-NS with tetR will bind to the specific site (tetO) desired and recruit wild type H- NS (without tetR) to bind to adjacent DNA non-specifically and silence them on transcription level as a result of the blockage of gene by both the mutated and wild type H-SN. We are using the green fluorescent protein (GFP) gene to test this principle. Normal genes without the binding of mutated H-SN with tetR to tetO site will have usual fluorescent intensity. However, if there is binding of mutated H-SN with tetR to the tetO site, there will be polymerisation of this specific DNA binding protein with the non-specific DNA binding protein. And hence, blocking of the transcription of the particular gene will result in silencing of gene. |
