Difference between revisions of "Part:BBa K2980011"

 
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__NOTOC__
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<partinfo>BBa_K2980011 short</partinfo>
  
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CRY2(R489E) is a mutant of Cryptochrome 2 (CRY2). By changing arginine on 489 to glutamate, the charge of C terminal is changed and the ability of CRY2 homo-oligomerization is lowered.
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===Background===
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In our system, phases are formed by CIB1-GCN(4)-FUS-GFP, while the recruitment of downstream proteins fused with mCherry-CRY2 into the phase is presented via the interaction between mCherry-CRY2 and CIB1-GCN(4)-FUS-GFP when stimulated. Therefore, without stimulation of 488nm laser, we anticipate that mCherry-CRY2 is smear in E.coli. Once stimulated, with strong interaction of CRY2-CIB1, mCherry-CRY2 is pulled into the phase. However, CRY2 homo-oligomerization is also stimulated by light 488nm, observed in our control experiment, which might alter the function of our system.
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===Design===
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To solve the problem, 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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{|class='wikitable'
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|'''Mutants'''
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|'''Principle'''
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|-
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|CRY2(R489E)
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|Change arginine on 489 to glutamate, making charge of C terminal negative.
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|-
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|CRY2(R489D)
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|Change arginine on 489 to aspartate, making charge of C terminal negative.
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|-
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|CRY2(Δ489-498)
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|Delete amino acids from 489-498, making charge of C terminal negative.
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|-
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|colspan=4|Table 1. Mutants of CRY2
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|-
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|}
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===Result===
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After observing under confocal, we found the extent of homo-oligomerization of CRY2 (R489E) is the lowest. Figure 1 shows confocal images of CRY2wt and CRY2(R489E).
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===Reference===
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[1] Yu, X., Liu, H., Klejnot, J., & Lin, C. (2010). The Cryptochrome Blue Light Receptors. ''The Arabidopsis Book'', 8(8). doi:10.1199/tab.0135
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[2] Engelhard, C., Wang, X., Robles, D., Moldt, J., Essen, L., Batschauer, A., ... & Ahmad, M. (2014). Cellular Metabolites Enhance the Light Sensitivity of Arabidopsis Cryptochrome through Alternate Electron Transfer Pathways. ''The Plant Cell'', 26(11), 4519-4531.
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===Sequence and Features===
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<partinfo>BBa_K2980000 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K2980000 parameters</partinfo>
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Revision as of 06:33, 16 October 2019

CRY2(R489E)

CRY2(R489E) is a mutant of Cryptochrome 2 (CRY2). By changing arginine on 489 to glutamate, the charge of C terminal is changed and the ability of CRY2 homo-oligomerization is lowered.


Background

In our system, phases are formed by CIB1-GCN(4)-FUS-GFP, while the recruitment of downstream proteins fused with mCherry-CRY2 into the phase is presented via the interaction between mCherry-CRY2 and CIB1-GCN(4)-FUS-GFP when stimulated. Therefore, without stimulation of 488nm laser, we anticipate that mCherry-CRY2 is smear in E.coli. Once stimulated, with strong interaction of CRY2-CIB1, mCherry-CRY2 is pulled into the phase. However, CRY2 homo-oligomerization is also stimulated by light 488nm, observed in our control experiment, which might alter the function of our system.

Design

To solve the problem, 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.

Mutants Principle
CRY2(R489E) Change arginine on 489 to glutamate, making charge of C terminal negative.
CRY2(R489D) Change arginine on 489 to aspartate, making charge of C terminal negative.
CRY2(Δ489-498) Delete amino acids from 489-498, making charge of C terminal negative.
Table 1. Mutants of CRY2

Result

After observing under confocal, we found the extent of homo-oligomerization of CRY2 (R489E) is the lowest. Figure 1 shows confocal images of CRY2wt and CRY2(R489E).


Reference

[1] Yu, X., Liu, H., Klejnot, J., & Lin, C. (2010). The Cryptochrome Blue Light Receptors. The Arabidopsis Book, 8(8). doi:10.1199/tab.0135

[2] Engelhard, C., Wang, X., Robles, D., Moldt, J., Essen, L., Batschauer, A., ... & Ahmad, M. (2014). Cellular Metabolites Enhance the Light Sensitivity of Arabidopsis Cryptochrome through Alternate Electron Transfer Pathways. The Plant Cell, 26(11), 4519-4531.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 393
    Illegal BglII site found at 852
    Illegal BamHI site found at 1331
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 277
    Illegal AgeI site found at 1006
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 629
    Illegal BsaI.rc site found at 38
    Illegal SapI.rc site found at 146