Difference between revisions of "Part:BBa K2980011:Design"

 
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===Design Notes===
 
===Design Notes===
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we introduce mutations on C terminal of CRY2, charges on which is probably reasonable for its oligomerization. According to theoretical postulation, positive charge on C terminal facilitates oligomerization while negative charge inhibits. Upon knowing that, the arginine on 489 is mutated to glutamate (R489E) and aspartate (R489D), or amino acids from 489 to 498 are completely deleted(Δ489-498), making the mutant C terminal more negative than wild type. Mutants and its principle is shown in Table1. CRY2wt and its mutants are all tagged with mCherry, in order to show the distribution of CRY2 in cells. Meanwhile, charge of N terminal is critical for CRY2-CIB1 interaction. We also change the N terminal charge of CRY2 to weak its interaction with CIB1 and increase light threshold of the system.
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We introduce mutations on C terminal of CRY2, charges on which is probably reasonable for its oligomerization. According to theoretical postulation, positive charge on C terminal facilitates oligomerization while negative charge inhibits. Upon knowing that, the arginine on 489 is mutated to glutamate (R489E) and aspartate (R489D), or amino acids from 489 to 498 are completely deleted(Δ489-498), making the mutant C terminal more negative than wild type. Mutants and its principle is shown in Table1. CRY2wt and its mutants are all tagged with mCherry, in order to show the distribution of CRY2 in cells. Meanwhile, charge of N terminal is critical for CRY2-CIB1 interaction. We also change the N terminal charge of CRY2 to weak its interaction with CIB1 and increase light threshold of the system.
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===Source===
 
===Source===

Revision as of 13:14, 15 October 2019


CRY2(R489E)


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 723
    Illegal PstI site found at 533
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 723
    Illegal PstI site found at 533
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 723
    Illegal BglII site found at 393
    Illegal BglII site found at 852
    Illegal BamHI site found at 1331
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 723
    Illegal PstI site found at 533
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 723
    Illegal PstI site found at 533
    Illegal AgeI site found at 277
    Illegal AgeI site found at 1006
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

We introduce mutations on C terminal of CRY2, charges on which is probably reasonable for its oligomerization. According to theoretical postulation, positive charge on C terminal facilitates oligomerization while negative charge inhibits. Upon knowing that, the arginine on 489 is mutated to glutamate (R489E) and aspartate (R489D), or amino acids from 489 to 498 are completely deleted(Δ489-498), making the mutant C terminal more negative than wild type. Mutants and its principle is shown in Table1. CRY2wt and its mutants are all tagged with mCherry, in order to show the distribution of CRY2 in cells. Meanwhile, charge of N terminal is critical for CRY2-CIB1 interaction. We also change the N terminal charge of CRY2 to weak its interaction with CIB1 and increase light threshold of the system.

Source

The part came from Prof. Pilong.Li's lab, an interlab collaboration

References