Difference between revisions of "Part:BBa K5301024"

Revision as of 14:14, 1 October 2024

Multi-polymer MSP, which refers to large circular MSPs through end-to-end connections of multiple MS

Usage and Biology

In order to produce large nanodiscs more conveniently, we hope to flexibly extend the length of MSP according to demand, and thus propose the concept of multi-polymer MSP, which refers to large circular MSPs through end-to-end connections of multiple MSP fragments. We used NW15 as the basic MSP and selected three types of linkers (Spy/Sdy/Snoop) to achieve the connection of different MSP fragments through the formation of covalent bonds, and adopted rigorous design to prevent self-cyclization of each fragment of the multi-polymer MSP. Finally, the successful cyclization of large circular MSPs is characterized by the fluorescence of mCherry after the combination of mCherry [1-10] and mCherry [11].

We also utilized AlphaFold 2 to simulate the overall structure of multi-polymerized MSP, and obtained multi-polymerized MSP with the correct conformation, as shown in the figure.

Figure 1.Alpha Fold 2 prediction model of the overall structure of multi-polymerized MSP

Characterization

We attempted to mix the three components of multi-polymerized MSP in vitro under two different conditions: first, by centrifuging the bacterial cultures, resuspending them, mixing the three bacterial suspensions, and then sonicating the mixture, followed by incubating the protein mixture at 4°C; alternatively, by mixing the three highly purified proteins after purification through nickel affinity chromatography. We verified the efficiency of the assembly of the three components through SDS-PAGE, as shown in the figure below.

From the figure, it can be observed that compared to the monomeric MSPs, the mixed product exhibits two new bands at approximately 100 and 180 kDa. These are presumed to be multi-polymerized MSP.

Figure 2.SDS-PAGE analysis of the construction of multi-polymerized MSP.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 3141
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 3141
Illegal NotI site found at 1720
Illegal NotI site found at 4038
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1239
Illegal BglII site found at 2323
Illegal BglII site found at 2695
Illegal BglII site found at 3557
Illegal BglII site found at 3929
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 3141
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 3141
Illegal AgeI site found at 1035
Illegal AgeI site found at 1828
Illegal AgeI site found at 2220
Illegal AgeI site found at 2980
1000
COMPATIBLE WITH RFC[1000]

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-===Sequence and Features===
+===Characterization===
+We attempted to mix the three components of multi-polymerized MSP in vitro under two different conditions: first, by centrifuging the bacterial cultures, resuspending them, mixing the three bacterial suspensions, and then sonicating the mixture, followed by incubating the protein mixture at 4°C; alternatively, by mixing the three highly purified proteins after purification through nickel affinity chromatography. We verified the efficiency of the assembly of the three components through SDS-PAGE, as shown in the figure below.
+<p>From the figure, it can be observed that compared to the monomeric MSPs, the mixed product exhibits two new bands at approximately 100 and 180 kDa. These are presumed to be multi-polymerized MSP.</p>
-<!-- Add more about the biology of this part here
+<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:min-content;"><div style="zoom:0.4;overflow:hidden;">
-===Usage and Biology===
+https://static.igem.wiki/teams/5301/parts/construction-of-tri.png
+</div><div class="thumbcaption">
+Figure 2.SDS-PAGE analysis of the construction of multi-polymerized MSP.
+</div></div></div></div>
+===Sequence and Features===
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