Team:SJTU-BioX-Shanghai/Project/Subproject1/Results 2
From 2011.igem.org
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We have tested luciferin reaction in cells with different combinations of tRNA Modulators and Reporters. We examined the changes in luciferase enzyme activity over time after rare tRNA expression is induced. The amount of luciferase is reflected indirectly by the bioluminescence emitted from the luciferin reaction. Results are shown below: | We have tested luciferin reaction in cells with different combinations of tRNA Modulators and Reporters. We examined the changes in luciferase enzyme activity over time after rare tRNA expression is induced. The amount of luciferase is reflected indirectly by the bioluminescence emitted from the luciferin reaction. Results are shown below: | ||
- | [[image:11-SJTU-compare-4.jpg|frame|center|Fig.1 (A)Enzyme activity of luciferase shown by bioluminescence emitted from the luciferin reaction reflecting the working curve of tRNA Modulator ''lacI''-Ptrc-tRNA<sup>Asp</sup> ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567001 BBa_K567001]) under Reporter P''bla''-Luc-4AGG([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567006 BBa_K567006]). (B)Enzyme activity of luciferase shown by bioluminescence emitted from the luciferin reaction reflecting the working curve of tRNA Modulator lacI-Ptrc-tRNA<sup>Asp</sup> ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567001 BBa_K567001]) under Reporter PT7-Luc-4AGG ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567009 BBa_K567009]). The working curve of tRNA Modulator ''lacI''-Ptrc-tRNA<sup>Asp</sup> fits typical titration curve.]] | + | [[image:11-SJTU-compare-4.jpg|frame|center|''Fig.1'' (A)Enzyme activity of luciferase shown by bioluminescence emitted from the luciferin reaction reflecting the working curve of tRNA Modulator ''lacI''-Ptrc-tRNA<sup>Asp</sup> ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567001 BBa_K567001]) under Reporter P''bla''-Luc-4AGG([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567006 BBa_K567006]). (B)Enzyme activity of luciferase shown by bioluminescence emitted from the luciferin reaction reflecting the working curve of tRNA Modulator lacI-Ptrc-tRNA<sup>Asp</sup> ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567001 BBa_K567001]) under Reporter PT7-Luc-4AGG ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567009 BBa_K567009]). The working curve of tRNA Modulator ''lacI''-Ptrc-tRNA<sup>Asp</sup> fits typical titration curve.]] |
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We examined the influence of different Reporter promoters on the working curve of tRNA Modulator, which is reflected by luciferase activity. Results showed that all the tRNA Modulator working curves fit titration curve, indicating that tRNA Modulator can act as a satisfying regulating tool. | We examined the influence of different Reporter promoters on the working curve of tRNA Modulator, which is reflected by luciferase activity. Results showed that all the tRNA Modulator working curves fit titration curve, indicating that tRNA Modulator can act as a satisfying regulating tool. | ||
- | [[image:11SJTUzhuzhuangtu-compare.jpg|frame|center|''Fig'' | + | [[image:11SJTUzhuzhuangtu-compare.jpg|frame|center|''Fig.3'' Enzyme activity of luciferase reaching plateau phase.]] |
However, the working range of tRNA Modulator is pre-defined by the strength of target protein promoter, T7 promoter and ''bla'' promoter in our project. | However, the working range of tRNA Modulator is pre-defined by the strength of target protein promoter, T7 promoter and ''bla'' promoter in our project. | ||
- | [[image:11sjtu_Compare_8.png|frame|center|''Fig'' | + | [[image:11sjtu_Compare_8.png|frame|center|''Fig.4'' (A) Working curve of tRNA Modulator lacI-Ptrc-tRNA<sup>Asp</sup> under Reporter P''bla''-Luc-8AGG reflected by bioluminescence emitted from the luciferin reaction. (B) Working curve of tRNA Modulator under Reporter PT7-Luc-8AGG. Here we analyze the influences of strong/weak promoter in the working curve of tRNA Modulator. |
Moreover, strong promoter (T7) of target gene can improve the titration curve of tRNA Modulator, indicating that tRNA Modulator works better under strong target protein promoters. ]] | Moreover, strong promoter (T7) of target gene can improve the titration curve of tRNA Modulator, indicating that tRNA Modulator works better under strong target protein promoters. ]] | ||
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The working curve of tRNA Modulator still fit titration curve under Reporters with different number of AGG insertions, indicating that the number of rare codons in the Reporter will not affect the stability and function of the Modulator. The influence of different number of rare codons in regulating protein biosynthesis is shown below: | The working curve of tRNA Modulator still fit titration curve under Reporters with different number of AGG insertions, indicating that the number of rare codons in the Reporter will not affect the stability and function of the Modulator. The influence of different number of rare codons in regulating protein biosynthesis is shown below: | ||
- | [[image:11sjtu_Compare_bla48_T72468.png|frame|center|''Fig.'' (A) Comparing the influence of different number of rare codon insertions in tRNA Modulator working curve. Two Reporters are compared, P''bla''-Luc-4AGG ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567005 BBa_K567005]) and P''bla''-Luc-8AGG ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567007 BBa_K567007]). (B) Comparing the influence of different number of rare codon insertions in tRNA Modulator working curve. Four Reporters are examined, including PT7-Luc-2AGG([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567008 BBa_K567008]), PT7-Luc-4AGG([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567009 BBa_K567009]), PT7-Luc-8AGG ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567010 BBa_K567010]) and PT7-Luc-6AGG ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567019 BBa_K567019]). ]] | + | [[image:11sjtu_Compare_bla48_T72468.png|frame|center|''Fig.5'' (A) Comparing the influence of different number of rare codon insertions in tRNA Modulator working curve. Two Reporters are compared, P''bla''-Luc-4AGG ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567005 BBa_K567005]) and P''bla''-Luc-8AGG ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567007 BBa_K567007]). (B) Comparing the influence of different number of rare codon insertions in tRNA Modulator working curve. Four Reporters are examined, including PT7-Luc-2AGG([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567008 BBa_K567008]), PT7-Luc-4AGG([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567009 BBa_K567009]), PT7-Luc-8AGG ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567010 BBa_K567010]) and PT7-Luc-6AGG ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567019 BBa_K567019]). ]] |
Revision as of 02:44, 6 October 2011