Difference between revisions of "Team:IIT-Madras/Demonstrations"
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The synthetic promoter library design is based on the bacteriophage T5 promoter with the intention of generating a series of promoters of varying expression rates. Some of these promoters have the standard iGEM RBS downstream of the promoter (the S series) whereas others have an optimal RBS as calculated by the Salis Lab RBS Calculator tool (the R series). In our constructs, these promoter-RBS sequences drive GFP expression in the pBAV1K vector in A. baylyi. The functioning of the promoter library is demonstrated by the observance of expression strengths differing from that of the original pBAV1K-T5-gfp construct (BioBrick Part BBaK1321309), which is used as the control. <br> | The synthetic promoter library design is based on the bacteriophage T5 promoter with the intention of generating a series of promoters of varying expression rates. Some of these promoters have the standard iGEM RBS downstream of the promoter (the S series) whereas others have an optimal RBS as calculated by the Salis Lab RBS Calculator tool (the R series). In our constructs, these promoter-RBS sequences drive GFP expression in the pBAV1K vector in A. baylyi. The functioning of the promoter library is demonstrated by the observance of expression strengths differing from that of the original pBAV1K-T5-gfp construct (BioBrick Part BBaK1321309), which is used as the control. <br> | ||
| − | <img src="https://static.igem.org/mediawiki/parts/9/96/T--IIT-Madras-Promoter-characterization.png"> | + | <img src="https://static.igem.org/mediawiki/parts/9/96/T--IIT-Madras-Promoter-characterization.png" style="width:100%"> |
The magnitudes of the fluorescent measurements are expressed here with respect to the fluorescence of the control. As seen in the graph, our promoters display a range of higher and lower expression rates. <br> | The magnitudes of the fluorescent measurements are expressed here with respect to the fluorescence of the control. As seen in the graph, our promoters display a range of higher and lower expression rates. <br> | ||
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Revision as of 00:28, 18 October 2018
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The synthetic promoter library design is based on the bacteriophage T5 promoter with the intention of generating a series of promoters of varying expression rates. Some of these promoters have the standard iGEM RBS downstream of the promoter (the S series) whereas others have an optimal RBS as calculated by the Salis Lab RBS Calculator tool (the R series). In our constructs, these promoter-RBS sequences drive GFP expression in the pBAV1K vector in A. baylyi. The functioning of the promoter library is demonstrated by the observance of expression strengths differing from that of the original pBAV1K-T5-gfp construct (BioBrick Part BBaK1321309), which is used as the control.
The magnitudes of the fluorescent measurements are expressed here with respect to the fluorescence of the control. As seen in the graph, our promoters display a range of higher and lower expression rates. The codon-bias table for A. baylyi generated using the ChassiDex CUTE tool was used to codon-optimize the eGFP and mCherry reporter proteins. Stronger expression of these reporters compared to the original pBAV1K-T5-gfp construct (BioBrick Part BBaK1321309) demonstrates that the codon-optimization process is functional.
As seen in the graph, the codon-optimized GFP shows significantly higher expression compared to the control.
References