Team:HKU-Hong Kong/Project

From 2011.igem.org

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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

@@ Line 3: / Line 3: @@
 |style="font-family: georgia, helvetica, arial, sans-serif;font-size:2em;color:#01DF01;"|Super Silencer
 |-
-|style="width:900px;"|'''Project Abstract'''
+|style="width:900px;"|'''Project Title'''
-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.
+'Super Silencer'---Development of a Novel Inducible Transcriptional Repressor mediates the formation of heterochromatin-like complex in ''E.coli''
-|style="width:900px;"|'''Project Overview'''
+'''Project Abstract'''
-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.
+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.
-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 H-NS. Fusion proteins with the H-NS domain were constructed and transformed into GFP-expressing E.coli. We engineered sites for the binding of these fusion proteins so that their binding can 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 H-NS domains and observed its effect in repressing GFP expression.
+'''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: