Difference between revisions of "Part:BBa K5301006"
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<partinfo>BBa_K5301006 short</partinfo>
 
<partinfo>BBa_K5301006 short</partinfo>
  
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===Usage and Biology===
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==Introduction==
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The goal of the BNU-China 2024 iGEM team is to fabricate nanodiscs, a kind of engineered nanoscale tool, by means of synthetic biology. Through literature review, we found MSP1E3D1 as the basic MSP element for constructing nanodiscs. We further sought and obtained spNW15 and spNW50 <ref>Zhang, S., et al., One-step construction of circularized nanodiscs using SpyCatcher-SpyTag. Nature Communications, 2021. 12(1): p. 5451.</ref>that utilized the automatic covalent linkage of SpyTag and SpyCatcher to enhance the cyclization efficiency and enable the automatic cyclization of MSP, in order to manufacture nanodiscs of different diameters more simply. On this basis, taking NW15 as the basic component, we designed the multi-polymerized MSP, consisting of three linear MSP monomers, providing a more flexible solution for manufacturing large nanodiscs, while reducing the expression pressure on the chassis bacteria and avoiding the difficulty of purifying large proteins.
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<p>This Part Collection aims to provide a series of easily accessible and distinctively characterized MSP proteins as a toolkit for the assembly of nanodiscs. Users can easily select which MSP to produce and utilize based on their own needs to manufacture nanodiscs. The nanodiscs fabricated using the MSP we designed can be used for stabilizing amphipathic proteins, studying the structure and function of amphipathic proteins, drug delivery, developing novel antiviral drugs, etc., and possess broad application prospects<ref>Padmanabha Das, K.M., et al., Large Nanodiscs: A Potential Game Changer in Structural Biology of Membrane Protein Complexes and Virus Entry. Frontiers in Bioengineering and Biotechnology, 2020. 8.</ref>.</p>
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<p>This part produces SnCSdT, as a part of the multi-polymerized MSP, to produce large nanodiscs more simply.</p>
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==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.  
 
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 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].

Revision as of 06:25, 2 October 2024


SnCSdT is one of the components of multi-polymerized MSP.

Introduction

The goal of the BNU-China 2024 iGEM team is to fabricate nanodiscs, a kind of engineered nanoscale tool, by means of synthetic biology. Through literature review, we found MSP1E3D1 as the basic MSP element for constructing nanodiscs. We further sought and obtained spNW15 and spNW50 [1]that utilized the automatic covalent linkage of SpyTag and SpyCatcher to enhance the cyclization efficiency and enable the automatic cyclization of MSP, in order to manufacture nanodiscs of different diameters more simply. On this basis, taking NW15 as the basic component, we designed the multi-polymerized MSP, consisting of three linear MSP monomers, providing a more flexible solution for manufacturing large nanodiscs, while reducing the expression pressure on the chassis bacteria and avoiding the difficulty of purifying large proteins.

This Part Collection aims to provide a series of easily accessible and distinctively characterized MSP proteins as a toolkit for the assembly of nanodiscs. Users can easily select which MSP to produce and utilize based on their own needs to manufacture nanodiscs. The nanodiscs fabricated using the MSP we designed can be used for stabilizing amphipathic proteins, studying the structure and function of amphipathic proteins, drug delivery, developing novel antiviral drugs, etc., and possess broad application prospects[2].

This part produces SnCSdT, as a part of the multi-polymerized MSP, to produce large nanodiscs more simply.

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]. SnCSdT, the second component of multi-polymerized MSP, is a fusion protein composed of SnoopCatcher, NW15, and SdyTag, with flexible GS linkers used to connect each part.

We also utilized AlphaFold 2 to simulate the structure of SnCSdT and obtained the correct linear, non-cyclized protein conformation, as shown in the figure.

tri-2-model.png

Figure 1.The structure of SnCSdT predicted by AlphaFold2


Plasmid Construction

To produce multi-polymerized MSP, we first constructed plasmids to synthesize three MSPs separately. We obtained the gene sequence of NW15 from NCBI and integrated the sequences of SnoopCatcher and SdyTag at its N-terminus and C-terminus to form SnCSdT. We added 5' (NcoI) and 3' (XhoI) to the ends of the gene through GENEWIZ, cloning them into the vector pET-28a(+) (Kanamycin) to construct three recombinant plasmids, which were then introduced into BL21 (DE3). Subsequently, we picked multiple single colonies from the plate for colony PCR and confirmed that the plasmids were successfully introduced into the host bacteria.

Cultivation, Purification and SDS-PAGE

Induction

We chose the T7 expression system as the pathway for protein expression and induced protein expression by adding IPTG at an appropriate time. Through experimental validation, we added the IPTG solution at a concentration of 0.8mM when the OD value of the bacterial suspension reached 0.6-0.8, resulting in substantial expression of soluble proteins.

tri-2-induction.png

Figure 2.SDS-PAGE analysis of SnCSdT protein with IPTG induction. An IPTG concentration of 0.8mM was used for 16 hours induction at 16°C. The molecular weight of SnCSdT is 42.1 kDa.

Purification

After confirming the successful expression of SnCSdT, we scaled up the culture and purified the protein. During plasmid construction, we incorporated a His-tag into the sequence, allowing for purification using nickel affinity chromatography, which specifically binds to His-tagged proteins. We eluted the protein using 300mM and 500mM imidazole, respectively, and obtained a large amount of target protein in both cases, as shown in figure 2.

tri-2-purification.png

Figure 3.SDS-PAGE Analysis of SnCSdT Purified by Nickel Affinity Chromatography. Both 300mM and 500mM imidazole elution resulted in the acquisition of a significant amount of target protein. The molecular weight of SnCSdT is 42.1 kDa.

Structure and biological activity analysis

We attempted to incubate three protein segments in vitro through different methods to link them, and successfully obtained mCherry fluorescence images.For more information on the construction of multi-polymerized MSP, go to BBa_K5301024.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1021
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1021
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 203
    Illegal BglII site found at 575
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1021
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1021
    Illegal AgeI site found at 100
    Illegal AgeI site found at 860
  • 1000
    COMPATIBLE WITH RFC[1000]


  1. ↑ Zhang, S., et al., One-step construction of circularized nanodiscs using SpyCatcher-SpyTag. Nature Communications, 2021. 12(1): p. 5451.
  2. ↑ Padmanabha Das, K.M., et al., Large Nanodiscs: A Potential Game Changer in Structural Biology of Membrane Protein Complexes and Virus Entry. Frontiers in Bioengineering and Biotechnology, 2020. 8.