Team:Hong Kong HKUST/pneumosensor/results/module two
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The three main components of the construct are <i>comX</i> gene, <i>comW</i> gene, and combox promoter. We assemble <i>comX</i> and combox promoter in one vector plasmid, while <i>comW</i> in a different plasmid. The system will be fused with a tagging protein and a reporting protein. Tagging protein is essential for detecting the ComX and ComW protein expression by means of western blot. Reporting protein which is fluorescence protein is needed for reporting purpose, hence <i>comX</i> and combox system could serve as a specific reporting system that will be useful for many synthetic constructs. <i>comX</i> and combox promoter construct will be assembled separately in different plasmid before being combined into one plasmid. </p> | The three main components of the construct are <i>comX</i> gene, <i>comW</i> gene, and combox promoter. We assemble <i>comX</i> and combox promoter in one vector plasmid, while <i>comW</i> in a different plasmid. The system will be fused with a tagging protein and a reporting protein. Tagging protein is essential for detecting the ComX and ComW protein expression by means of western blot. Reporting protein which is fluorescence protein is needed for reporting purpose, hence <i>comX</i> and combox system could serve as a specific reporting system that will be useful for many synthetic constructs. <i>comX</i> and combox promoter construct will be assembled separately in different plasmid before being combined into one plasmid. </p> | ||
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Revision as of 14:54, 5 October 2014
Pneumosensor Results
S. pneumoniae sigma X promoters Module
Overview
The activity of combox promoter is turned on by a specific sigma factor that is produced by a regulatory gene comX. The sigma factor X will bind to the combox promoter region and activate gene expression. Sigma factor X will serve as an inducer with high specificity as it binds to an area of several specific base pairs on the combox promoter. This ComX and combox system could be used as a highly specific reporting system in our Streptococcus pneumonia detection platform.
However in nature, ComX protein will be degraded by clpXP enzyme which exist in E.Coli and some other bacteria. Hence, to ensure the induction of combox promoter by comX, comW protein is needed as it functions to protect ComX protein from being degraded by clpXP. ComW protein will be degraded instead, increasing the amount of ComX protein produced.
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ComX Generator construct (BBa_K1379006) and comW construct
Backbone pSB1C3 was used for ComX generator construct and comW construct. comX gene / comW gene was fused with BBa_K880005 which contains a constitutive promoter (J23100) and strong RBS (B0032). The purpose of this strong constitutive promoter and strong RBS is to unsure the large production of ComX and ComW protein throughout time. Then, a double terminator (B0015) is fused with the promoter, RBS, and ComX. BBa_K880005 and B0015 was obtained from 2014 iGEM distribution kit.
comX Tag Protein |
PCelA (BBa_ K1379002) and Phelicase (BBa_ K1379003) construct
Backbone pSB1C3 was used for PCelA and Phelicase construct. PCelA / Phelicase gene was fused with BBa_E0240, which contains a medium RBS (BBa_B0032), GFP (BBa_E0040) and double terminator (BBa_B0015). The purpose of this GFP generator is to indicate the functionality of PCelA and Phelicase in the presence and absence of ComX protein. BBa_E0240 was obtained from 2014 iGEM distribution kit. The bacterial strain of E.coli used is DH10B.
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Assembly and Characterization
Assembly |
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