Team:HKU-Hong Kong/Project

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Latest revision as of 14:12, 5 October 2011

Super Silencer

Project Title

'Super Silencer'---Development of a Novel Inducible Transcriptional Repressor mediates the formation of heterochromatin-like complex in E.coli

Project Abstract

In eukaryotes, heterochromatin plays an important role in gene regulation. Here we use a synthetic biology approach to imitate heterochromatin in E.coli to achieve gene silencing. Specifically, fusion proteins comprising tetR and different parts of HNS (histone-like nucleoid structuring protein) were synthesized, they are expected to bind DNA specifically and carry out polymerization among the fusion HNS and the native HNS to create a densely packed DNA form, which may block the transcription. We produced constructs with tetO sites upstream or downstream of lac promoter and EGFP gene. Then we used standard constitutive promoters with different activity to drive our fusion proteins to find the optimum expression level. Moreover, tetR, HNS and fusion proteins were purified and gel shift assay would be utilized to detect the interaction between those proteins with DNA. This study presents a novel approach to introduce a mimic heterochromatin-like structure into prokaryotes to achieve inducible gene silencing.

Project Overview

Gene silencing is not observed in bacteria in nature; however, the ability to knockdown the transcription of specific genes promises an attractive tool for bacterial reverse genetics, not to mention further possibilities of ‘silencing’ toxin-encoding genes in many pathogenic bacteria.

It is therefore of our interest to mimic this phenomena in E.coli by developing an ‘alternate’ mechanism using synthetic biology approaches. Our project attempts to test this by making use of the gene-repressing properties of a naturally-occurring protein in many gram-negative bacteria: the nucleoid protein HNS. Fusion proteins with the HNS domain were constructed and transformed into GFP-expressing E.coli. We engineered sites for the binding of these fusion proteins so that their binding could facilitate the repression of the GFP gene. To optimize the repression effect, we engineered the sites at different locations and also constructed fusion proteins with different HNS domains and observed its effect in repressing GFP expression.

In this project we:

Created a total of eight BioBricks
Showed the possibility of silencing specific genes using fusion protein constructs and engineered DNA sequences

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-|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#01DF01;"|Super Silencer
+|style="font-family: georgia, helvetica, arial, sans-serif;font-size:2em;color:#01DF01;"|Super Silencer
 |-
-|style="width:900px;"|'''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
+|style="width:900px;"|'''Project Title'''
-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.
+'Super Silencer'---Development of a Novel Inducible Transcriptional Repressor mediates the formation of heterochromatin-like complex in ''E.coli''
-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.'''
+'''Project Abstract'''
+In eukaryotes, heterochromatin plays an important role in gene regulation. Here we use a synthetic biology approach to imitate heterochromatin in ''E.coli'' to achieve gene silencing. Specifically, fusion proteins comprising tetR and different parts of HNS (histone-like nucleoid structuring protein) were synthesized, they are expected to bind DNA specifically and carry out polymerization among the fusion HNS and the native HNS to create a densely packed DNA form, which may block the transcription. We produced constructs with tetO sites upstream or downstream of lac promoter and EGFP gene. Then we used standard constitutive promoters with different activity to drive our fusion proteins to find the optimum expression level. Moreover, tetR, HNS and fusion proteins were purified and gel shift assay would be utilized to detect the interaction between those proteins with DNA. This study presents a novel approach to introduce a mimic heterochromatin-like structure into prokaryotes to achieve inducible gene silencing.
+'''Project Overview'''
+Gene silencing is not observed in bacteria in nature; however, the ability to knockdown the transcription of specific genes promises an attractive tool for bacterial reverse genetics, not to mention further possibilities of ‘silencing’ toxin-encoding genes in many pathogenic bacteria.
+It is therefore of our interest to mimic this phenomena in ''E.coli'' by developing an ‘alternate’ mechanism using synthetic biology approaches. Our project attempts to test this by making use of the gene-repressing properties of a naturally-occurring protein in many gram-negative bacteria: the nucleoid protein HNS. Fusion proteins with the HNS domain were constructed and transformed into GFP-expressing E.coli. We engineered sites for the binding of these fusion proteins so that their binding could facilitate the repression of the GFP gene. To optimize the repression effect, we engineered the sites at different locations and also constructed fusion proteins with different HNS domains and observed its effect in repressing GFP expression.
+In this project we:
+<OL>
+<LI>Created a total of eight BioBricks
+<LI>Showed the possibility of silencing specific genes using fusion protein constructs and engineered DNA sequences