Team:SJTU-BioX-Shanghai/Project/Subproject1-3
From 2011.igem.org
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[[image:11sjtu_Compare_8.png|frame|center|''Fig.3'' (A) Luciferase (P''bla''-Luc-8AGG) production in cells overexpressing rare tRNA with ''lacI''-Ptrc-tRNA<sup>Arg</sup>. Luciferase production is reflected by bioluminescence emitted from the luciferin reaction. (B) Luciferase production in cells with PT7-Luc-8AGG as reporter. Here we analyze the influences of strong/weak promoter in luciferase production. Strong promoter (T7) of target gene can improve the titration curve, indicating that our device works better under strong target protein promoters. ]] | [[image:11sjtu_Compare_8.png|frame|center|''Fig.3'' (A) Luciferase (P''bla''-Luc-8AGG) production in cells overexpressing rare tRNA with ''lacI''-Ptrc-tRNA<sup>Arg</sup>. Luciferase production is reflected by bioluminescence emitted from the luciferin reaction. (B) Luciferase production in cells with PT7-Luc-8AGG as reporter. Here we analyze the influences of strong/weak promoter in luciferase production. Strong promoter (T7) of target gene can improve the titration curve, indicating that our device works better under strong target protein promoters. ]] | ||
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==Location of Rare Codons== | ==Location of Rare Codons== | ||
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[[image:11SJTU_rare_26.jpg|frame|center]] | [[image:11SJTU_rare_26.jpg|frame|center]] | ||
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+ | ===Note: Our device can be used as a regulating tool=== | ||
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+ | We have tested luciferin reaction in cells. 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: | ||
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+ | [[image:11-SJTU-compare-4.jpg|frame|center|''Fig.4'' (A)Luciferase (P''bla''-Luc-4AGG([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567006 BBa_K567006])) production reflected by bioluminescence emitted from the luciferin reaction. (B)Luciferase (PT7-Luc-4AGG ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567009 BBa_K567009])) production reflected by bioluminescence emitted from the luciferin reaction. ]] | ||
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+ | Here we use the above two curves as examples to characterize the working curve of our device. Both curves fits typical titration curve, indicating that our device can function as a regulating tool. | ||
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+ | The rest of the working curves are shown here: | ||
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+ | <gallery caption="Pbla"> | ||
+ | image:11SJTUResultPlbla6.jpg|''Fig.5'' | ||
+ | image:11SJTUResultPbla8.jpg|''Fig.6'' | ||
+ | </gallery> | ||
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+ | <gallery caption="PT7"> | ||
+ | image:11SJTUT7-2.png|''Fig.7'' | ||
+ | image:11SJTUT7-6.png|''Fig.8'' | ||
+ | image:11SJTUT7-8.png|''Fig.9'' | ||
+ | </gallery> | ||
+ | '''Note''':Click to see large figures. | ||
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+ | Results showed that all the devices’ working curves fit titration curve, indicating that our device can act as a satisfying regulating tool. | ||
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+ | From this experiment, we noticed that the typical working curve of our device can be better observed under IPTG induced ''lacI''-Ptrc-tRNA<sup>Arg</sup> ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567001 BBa_K567001]) compared with UV excitation induced sulA promoter-tRNA<sup>Arg</sup>([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567002 BBa_K567002]), though sulA promoter-tRNA<sup>Arg</sup> responded quicker to signals. So in the above experiments, we test with ''lacI''-Ptrc-tRNA<sup>Arg</sup>. |
Revision as of 01:37, 29 October 2011