Difference between revisions of "Part:BBa K2036023"
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This part is built by In-Fusion cloning method and together with [https://parts.igem.org/Part:BBa_K2036021 BBa_K2036021] and [https://parts.igem.org/Part:BBa_K2036023 BBa_K2036023] to further assemble [https://parts.igem.org/Part:BBa_K2036024 BBa_K2036024]. pRM can be negatively regulated by Cro. ilDH and beta-glactosidase are important enzyme in celluler metobolism.
 
This part is built by In-Fusion cloning method and together with [https://parts.igem.org/Part:BBa_K2036021 BBa_K2036021] and [https://parts.igem.org/Part:BBa_K2036023 BBa_K2036023] to further assemble [https://parts.igem.org/Part:BBa_K2036024 BBa_K2036024]. pRM can be negatively regulated by Cro. ilDH and beta-glactosidase are important enzyme in celluler metobolism.
 
[[File:T--HUST-China--Experiments-Fig15.png|thumb|350px|center|Fig1: Prokaryote application plasmid for lactose intolerance]]
 
[[File:T--HUST-China--Experiments-Fig15.png|thumb|350px|center|Fig1: Prokaryote application plasmid for lactose intolerance]]
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<p>
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We characterized cro and pRM inhibition by the same method as CI and pR’s. From line chart and fluorescence detection, we can see that the test group contains cro expressed less GFP protein than control group over time. It proves that cro can effectively bind pRM to block its downstream gene’s transcription.
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</p>
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[[File: T--HUST-China--CI-pR_inhibition.png |thumb|800px|center|Fig: Cro and pRM inhibition test]]
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<h3> Beta-galactosidase activity:</h3>
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<p>
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Due to the limited time before wiki freezing, we didn’t completed the test of lactic balance function of our engineered strain in vitro. But we tried to characterize pH-induced beta-galactosidase’s expression level to prove that half of our application circuit (BBa_K2036024)works.
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We tested enzyme activity of our strain cultured at pH6.5, 7.5 and 8.5.
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</p>
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[[File: T--HUST-China--enzyme-activity.png |thumb|800px|center|Fig: Beta-galactosidase activity]]
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<p>
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As the data shows, beta-galactosidase activity of our strain cultured at pH8.5 was significantly higher than the other two groups which is corresponding to our expectations: When pH comes back to 7~9, our strain will sense the change and express beta-galactosidase.
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</p>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 17:23, 19 October 2016


RBS-iLDH-TT-pRM-RBS-beta-galactosidase

This part is built by In-Fusion cloning method and together with BBa_K2036021 and BBa_K2036023 to further assemble BBa_K2036024. pRM can be negatively regulated by Cro. ilDH and beta-glactosidase are important enzyme in celluler metobolism.

Fig1: Prokaryote application plasmid for lactose intolerance


We characterized cro and pRM inhibition by the same method as CI and pR’s. From line chart and fluorescence detection, we can see that the test group contains cro expressed less GFP protein than control group over time. It proves that cro can effectively bind pRM to block its downstream gene’s transcription.

Fig: Cro and pRM inhibition test


Beta-galactosidase activity:

Due to the limited time before wiki freezing, we didn’t completed the test of lactic balance function of our engineered strain in vitro. But we tried to characterize pH-induced beta-galactosidase’s expression level to prove that half of our application circuit (BBa_K2036024)works. We tested enzyme activity of our strain cultured at pH6.5, 7.5 and 8.5.

Fig: Beta-galactosidase activity

As the data shows, beta-galactosidase activity of our strain cultured at pH8.5 was significantly higher than the other two groups which is corresponding to our expectations: When pH comes back to 7~9, our strain will sense the change and express beta-galactosidase.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 737
    Illegal BamHI site found at 776
    Illegal BamHI site found at 1468
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]