Difference between revisions of "Part:BBa K2980011"

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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).

Figure 1A Confocal image of CRY2wt (Zoom 1), most E.coli form phases, showing high ability of oligomerization
Figure 1B Confocal image of CRY2 (R489E)(Zoom 1), mCherry in E.coli are smear, showing low abilityof oligomerization.

All samples are induced with 0.05mM IPTG for 2 h at 16℃. Figure 2 shows homo-oligomerization details of CRY2wt and CRY2(R489E).

Figure 2A Confocal image of CRY2wt(Zoom 3), CRY2wt forms 2 phases on both ends of E.coli
Figure 2B Confocal image of CRY2(R489E)(Zoom 3), CRY2(R489E) is almost smear in E.coli.

Although in some cells phase is formed, the phase is much more smaller and darker than the phase formed by CRY2wt. All samples are induced with 0.05mM IPTG for 2 h at 16℃ We carried out statistical analysis after acquiring merge images (mCherry channel and TD). Three kinds of objects are count in our observation: bacteria with phase separation, bacteria without phase separation, bacteria whose state was unclear (probably because of low expression level for unknown reason or too young to accumulate enough proteins). The examples of captured pictures were given in Figure 3. Then, the total number of bacteria was added up. Finally, we used the ratio of group “phase” to sum to indicate the ability of CRY2 oligomerization under the same condition. The distributions of the data are showed in Figure 4.

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

@@ Line 32: / Line 32: @@
 <li style="display: inline-block;"> [[File:2019 THU CRY2(R489E) Figure 1B.png|thumb|none|400px|Figure 1B Confocal image of CRY2 (R489E)(Zoom 1), mCherry in E.coli are smear, showing low abilityof oligomerization.]]</li>
 </ul></div>
+All samples are induced with 0.05mM IPTG for 2 h at 16℃.
+Figure 2 shows homo-oligomerization details of CRY2wt and CRY2(R489E).
+<div><ul>
+<li style="display: inline-block;">[[File:2019 THU CRY2(R489E) Figure 2A.png|thumb|none|400px|Figure 2A Confocal image of CRY2wt(Zoom 3), CRY2wt forms 2 phases on both ends of E.coli
+ ]] </li>
+<li style="display: inline-block;"> [[File:2019 THU CRY2(R489E) Figure 2B.png|thumb|none|400px|Figure 2B Confocal image of CRY2(R489E)(Zoom 3), CRY2(R489E) is almost smear in E.coli. ]]</li>
+</ul></div>
+Although in some cells phase is formed, the phase is much more smaller and darker than the phase formed by CRY2wt. All samples are induced with 0.05mM IPTG for 2 h at 16℃
+We carried out statistical analysis after acquiring merge images (mCherry channel and TD). Three kinds of objects are count in our observation: bacteria with phase separation, bacteria without phase separation, bacteria whose state was unclear (probably because of low expression level for unknown reason or too young to accumulate enough proteins). The examples of captured pictures were given in Figure 3. Then, the total number of bacteria was added up. Finally, we used the ratio of group “phase” to sum to indicate the ability of CRY2 oligomerization under the same condition. The distributions of the data are showed in Figure 4.
 ===Reference===