Difference between revisions of "Part:BBa K5301024"

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Figure 2.SDS-PAGE analysis of the construction of multi-polymerized MSP.  
 
Figure 2.SDS-PAGE analysis of the construction of multi-polymerized MSP.  
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===Structure and biological activity analysis===
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Since the results from SDS-PAGE did not provide us with reliable data, we aimed to characterize the successful construction of the multi-polymerized MSP through the observation of mCherry fluorescence. We used a fluorescence inverted microscope to observe the mixture obtained from the first method mentioned above and found red fluorescence under green light excitation, thus confirming the successful ligation of the mCherry protein. Based on this, we could indicate that we have successfully constructed the multi-polymerized MSP.
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Figure 3.The mCherry fluorescence observation chart (10×10) under green light excitation. It was observed using a fluorescent Inverted microscope and photographed with an ordinary mobile phone.
 
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Revision as of 14:36, 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.

msp1.gif

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, additionally, the obtained mixture was concentrated to make the target bands more prominent; 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.

construction-of-tri.png

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

Structure and biological activity analysis

Since the results from SDS-PAGE did not provide us with reliable data, we aimed to characterize the successful construction of the multi-polymerized MSP through the observation of mCherry fluorescence. We used a fluorescence inverted microscope to observe the mixture obtained from the first method mentioned above and found red fluorescence under green light excitation, thus confirming the successful ligation of the mCherry protein. Based on this, we could indicate that we have successfully constructed the multi-polymerized MSP.

yingguang.png

Figure 3.The mCherry fluorescence observation chart (10×10) under green light excitation. It was observed using a fluorescent Inverted microscope and photographed with an ordinary mobile phone.

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]