Difference between revisions of "Part:BBa K5301005"

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<partinfo>BBa_K5301005 short</partinfo>
 
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===Usage and Biology===
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==Introduction==
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The goal of BNU-China 2024 iGEM team is to fabricate nanodiscs, a kind of engineered nanoscale tool, by means of synthetic biology. Our parts collection can be mainly divided into two categories: mono-MSPs that could construct small or large nanodiscs through self-cyclization, and large cyclic MSP formed by the interaction and linkage of multiple MSPs, which are used for constructing large nanodiscs. They are closely linked together due to their common function of manufacturing nanodiscs.
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<p>Through literature review, we found MSP1E3D1 as the basic MSP element for constructing nanodiscs<ref>Ilia G. Denisov, Bradley J. Baas, Yelena V. Grinkova, Stephen G. Sligar, Cooperativity in Cytochrome P450 3A4: LINKAGES IN SUBSTRATE BINDING, SPIN STATE, UNCOUPLING, AND PRODUCT FORMATION*, Journal of Biological Chemistry, Volume 282, Issue 10, 2007, Pages 7066-7076, ISSN 0021-9258, https://doi.org/10.1074/jbc.M609589200.</ref>. 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. Only when three mono-MSPs interact with each other can they form cyclized MSP and achieve their function of constructing nanodiscs. It provides 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. </p>
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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 SCSdC-mCh[1-10], 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].
 
SCSdC-mCh[1-10], the first component of multi-polymerized MSP, is a fusion protein composed of SpyCatcher, mCherry [1-10], NW15, and SdyCatcher, with flexible GS linkers used to connect each part.
 
SCSdC-mCh[1-10], the first component of multi-polymerized MSP, is a fusion protein composed of SpyCatcher, mCherry [1-10], NW15, and SdyCatcher, with flexible GS linkers used to connect each part.
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<p>We also utilized AlphaFold 2 to simulate the structure of SCSdC-mCh[1-10] and obtained the correct linear, non-cyclized protein conformation, as shown in the figure. </p>
  
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==Cultivation, Purification and SDS-PAGE==
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===Induction===
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https://static.igem.wiki/teams/5301/parts/tri-1-model.png
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Figure 1.The structure of SCSdC-mCh[1-10] predicted by AlphaFold2
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==Plasmid Construction==
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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 SpyCatcher and SdyCatcher at its N-terminus and C-terminus to form SCSdC-mCh[1-10]. 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 to check if the plasmids were successfully introduced into the host bacteria.
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Figure 2.Colony PCR Results of Host Bacteria Introduced with pET28a_SCSdC-mCh[1-10], All Four Colonies Successfully Obtained the Desired Band, Indicating Successful Plasmid Transformation.The theoretical sequence length is 2095 bp.
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==Cultivation, Purification and SDS-PAGE==
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===Induction===
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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.
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https://static.igem.wiki/teams/5301/parts/tri-1-induction.png
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Figure 3.SDS-PAGE analysis of SCSdC-mCh[1-10] protein with IPTG induction. An IPTG concentration of 0.8mM was used for 16 hours induction at 16°C. The molecular weight of SCSdC-mCh[1-10] is 76.3 kDa.
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===Purification===
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After confirming the successful expression of SCSdC-mCh[1-10], 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 4.
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https://static.igem.wiki/teams/5301/parts/tri-1-purification.png
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Figure 4.SDS-PAGE Analysis of SCSdC-mCh[1-10] 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 SCSdC-mCh[1-10] is 76.3 kDa.
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===Further Purification by SEC===
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Due to various possible reasons, the target protein obtained through nickel affinity chromatography has a relatively high concentration but still contains a significant amount of impurity proteins, which is detrimental to subsequent characterization experiments. Therefore, we chose to further purify the target protein to a higher degree of purity through SEC (Size Exclusion Chromatography). According to Figure 5, we obtained a total of six elution peaks. Through SDS-PAGE analysis of the samples corresponding to each peak, we confirmed that the purified target protein was obtained at the second and third peaks.
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Figure 5.SEC absorption peak chromatogram of the purification results of SCSdC-mCh[1-10].
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Figure 6.SDS-PAGE Analysis of SCSdC-mCh[1-10] After Further Purification by SEC. The third and fourth lanes show the presence of the target protein (corresponding to elution volumes of 56.3 and 64.55, respectively). The molecular weight of SCSdC-mCh[1-10] is 76.3 kDa.
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    <figcaption>Figure 1 | SDS-PAGE analysis of SCSdC-mCh[1-10] protein with IPTG concentration gradient induction. An IPTG concentration of 0.8mM was used for 16 hours induction at 16°C.</figcaption>
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    <p>Basic Silinker (BS) is a novel recombinant protein that efficiently attaches to the surface of silicon dioxide. The presence of mSA often leads to the formation of inclusion bodies, increasing the difficulty in purification. To achieve efficient expression of Basic Silinker and minimize the formation of inclusion bodies, we screened the induction conditions using IPTG. We set up a gradient of five IPTG concentrations: 0mM, 0.1mM, 0.25mM, 0.5mM, and 1mM. The results showed that the optimal protein expression was achieved with a concentration of 1mM. Furthermore, we tested two temperature gradients for induction: 37°C and 16°C. At 37°C, the protein mainly formed inclusion bodies rather than soluble proteins. Therefore, we determined that 16°C was the effective induction temperature.
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To facilitate proper folding of mSA and reduce inclusion body formation, we modified the protein buffer by incorporating biotin. The binding of biotin to mSA helps with the correct folding of the Basic Silinker protein, minimizing the formation of misfolded inclusion bodies. As a result, we obtained soluble proteins that could be extracted from the supernatant. The formulation of the buffer and experimental procedures can be found in our protocol.</p>
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==Structure and biological activity analysis==
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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 <partinfo>BBa_K5301024</partinfo>.
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===Sequence and Features===
  
 
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<partinfo>BBa_K5301005 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5301005 SequenceAndFeatures</partinfo>
  

Latest revision as of 10:09, 2 October 2024


SCSdC-mCh[1-10] is one of the components of multi-polymerized MSP.

Introduction

The goal of BNU-China 2024 iGEM team is to fabricate nanodiscs, a kind of engineered nanoscale tool, by means of synthetic biology. Our parts collection can be mainly divided into two categories: mono-MSPs that could construct small or large nanodiscs through self-cyclization, and large cyclic MSP formed by the interaction and linkage of multiple MSPs, which are used for constructing large nanodiscs. They are closely linked together due to their common function of manufacturing nanodiscs.

Through literature review, we found MSP1E3D1 as the basic MSP element for constructing nanodiscs[1]. We further sought and obtained spNW15 and spNW50 [2]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. Only when three mono-MSPs interact with each other can they form cyclized MSP and achieve their function of constructing nanodiscs. It provides 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[3].

This part produces SCSdC-mCh[1-10], 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]. SCSdC-mCh[1-10], the first component of multi-polymerized MSP, is a fusion protein composed of SpyCatcher, mCherry [1-10], NW15, and SdyCatcher, with flexible GS linkers used to connect each part.

We also utilized AlphaFold 2 to simulate the structure of SCSdC-mCh[1-10] and obtained the correct linear, non-cyclized protein conformation, as shown in the figure.

tri-1-model.png

Figure 1.The structure of SCSdC-mCh[1-10] 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 SpyCatcher and SdyCatcher at its N-terminus and C-terminus to form SCSdC-mCh[1-10]. 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 to check if the plasmids were successfully introduced into the host bacteria.

tri-1-pcr.png

Figure 2.Colony PCR Results of Host Bacteria Introduced with pET28a_SCSdC-mCh[1-10], All Four Colonies Successfully Obtained the Desired Band, Indicating Successful Plasmid Transformation.The theoretical sequence length is 2095 bp.

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-1-induction.png

Figure 3.SDS-PAGE analysis of SCSdC-mCh[1-10] protein with IPTG induction. An IPTG concentration of 0.8mM was used for 16 hours induction at 16°C. The molecular weight of SCSdC-mCh[1-10] is 76.3 kDa.

Purification

After confirming the successful expression of SCSdC-mCh[1-10], 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 4.

tri-1-purification.png

Figure 4.SDS-PAGE Analysis of SCSdC-mCh[1-10] 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 SCSdC-mCh[1-10] is 76.3 kDa.

Further Purification by SEC

Due to various possible reasons, the target protein obtained through nickel affinity chromatography has a relatively high concentration but still contains a significant amount of impurity proteins, which is detrimental to subsequent characterization experiments. Therefore, we chose to further purify the target protein to a higher degree of purity through SEC (Size Exclusion Chromatography). According to Figure 5, we obtained a total of six elution peaks. Through SDS-PAGE analysis of the samples corresponding to each peak, we confirmed that the purified target protein was obtained at the second and third peaks.

tri-1-sec.jpg

Figure 5.SEC absorption peak chromatogram of the purification results of SCSdC-mCh[1-10].

tri-1-sec-result.png

Figure 6.SDS-PAGE Analysis of SCSdC-mCh[1-10] After Further Purification by SEC. The third and fourth lanes show the presence of the target protein (corresponding to elution volumes of 56.3 and 64.55, respectively). The molecular weight of SCSdC-mCh[1-10] is 76.3 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
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 1720
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1239
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1035
    Illegal AgeI site found at 1828
  • 1000
    COMPATIBLE WITH RFC[1000]


  1. ↑ Ilia G. Denisov, Bradley J. Baas, Yelena V. Grinkova, Stephen G. Sligar, Cooperativity in Cytochrome P450 3A4: LINKAGES IN SUBSTRATE BINDING, SPIN STATE, UNCOUPLING, AND PRODUCT FORMATION*, Journal of Biological Chemistry, Volume 282, Issue 10, 2007, Pages 7066-7076, ISSN 0021-9258, https://doi.org/10.1074/jbc.M609589200.
  2. ↑ Zhang, S., et al., One-step construction of circularized nanodiscs using SpyCatcher-SpyTag. Nature Communications, 2021. 12(1): p. 5451.
  3. ↑ 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.