Parts Collection
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 (BBa_K5301000) as the basic MSP element for constructing nanodiscs [1]. We further sought and obtained spNW15 (BBa_K5301013) and spNW50 (BBa_K5301015) [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 (BBa_K5301024), consisting of three linear MSP monomers (BBa_K5301005, BBa_K5301006, BBa_K5301007). 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].
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BBa_K5301000 - Produces MSP1E3D1, fabricating nanodiscs with relatively high lipid fluidity [4].
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BBa_K5301013 - Produces spNW15, manufacturing small-diameter and easily cyclized nanodiscs, and can be flexibly modified to produce larger nanodiscs.
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BBa_K5301015 - Produces spNW50, manufacturing large-diameter and easily cyclized nanodiscs.
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BBa_K5301005 - Produces SCSdC-mCh[1-10], as a part of the multi-polymerized MSP, to produce large nanodiscs more simply.
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BBa_K5301006 - Produces SnCSdT, as a part of the multi-polymerized MSP, to produce large nanodiscs more simply.
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BBa_K5301007 - Produces SnTST-mCh[11], as a part of the multi-polymerized MSP, to produce large nanodiscs more simply.
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BBa_K5301024 - The multi-polymerized MSP is a large-sized cyclized MSP that connects three MSP parts through different linkers, facilitating the manufacture of larger nanodiscs.
Basic Parts
| Number | Name | Description |
|---|---|---|
| BBa_K2572009 | lacI promoter | Initiating the expression of lacI gene. |
| BBa_K1088018 | lacI | It binds to lac operator to inhibit transcription in E. coli, which could be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG). |
| BBa_K3633015 | T7 promoter | A common promoter that can be induced by IPTG. We use the promoter to produce many types of MSPs and some other proteins like GFP. |
| BBa_K3286004 | lac operator | The lac repressor binds to it to inhibit transcription in E. coli, which could be relieved by adding lactose or IPTG. |
| BBa_K731721 | T7 terminator | A wild type terminator from T7 bacteriophage. |
| BBa_I712004 | CMV promoter | A constitutive expression promoter used in our capsid plasmid for expression of PEG10 protein. |
| BBa_K4140013 | VSVg-Fusogen | A typical viral fusion protein that can bind to LDL receptors and trigger membrane fusion. |
| BBa_K4140022 | PEG10 | A protein that binds its own mRNA and self-assembles into virion-like extracellular vesicles. |
| BBa_K5301016 | PEG10 UTR flanked-GFP | The mRNA produced can be carried by the VLP formed by PEG10. |
| BBa_K4623001 | monomeric streptavidin(mSA) | Streptavidin is a tetramer protein that binds to tetramolecular biotin with high specificity. |
| BBa_K5301012 | sGFP1-10 | The first 10 β-strands of the GFP β-barrel can emit fluorescence upon binding with sGFP11. |
| BBa_K5301014 | sGFP11 | The last β-strand of the GFP β-barrel can emit fluorescence upon binding with sGFP1-10. |
| BBa_K5301017 | sGFP1-10 tether | Used for constructing dimeric membrane proteins. |
| BBa_K5301018 | sGFP11 tether | Used for constructing dimeric membrane proteins. |
| BBa_K5301011 | spMSP1D1 | It expresses the SpyCatcher-MSP1D1-SpyTag fusion protein for constructing nanodiscs. |
| BBa_K5301000 | MSP1E3D1 | MSP1E3D1 could construct nanodiscs with a diameter of 12.9 nm. |
| BBa_K5301013 | spNW15 | It is the truncated form of spNW30, capable of constructing nanodiscs with a diameter of 15nm. |
| BBa_K5301015 | spNW50 | NW50 is a high molecular weight membrane scaffold protein. |
| BBa_K5301004 | SpyCatcher | It can cooperate with SpyTag for covalent binding of proteins. |
| BBa_K5301003 | SpyTag | It can cooperate with SpyCatcher for covalent binding of proteins. |
| BBa_K5301001 | SdyCatcher | It can cooperate with SdyTag for covalent binding of proteins. |
| BBa_K5301002 | SdyTag | It can cooperate with SdyCatcher for covalent binding of proteins. |
| BBa_K4247009 | SnoopCatcher | It can cooperate with SnoopTag for covalent binding of proteins. |
| BBa_K4247008 | SnoopTag | It can cooperate with SnoopCatcher for covalent binding of proteins. |
| BBa_K5301008 | mCherry1-10 | Used as a bimolecular fluorescence complementation system. |
| BBa_K5301009 | mCherry11 | Used as a bimolecular fluorescence complementation system. |
| BBa_K5301005 | SCSdC-mCh[1-10] | It is the first component of multi-polymerized MSP. |
| BBa_K5301006 | SnCSdT | It is the second component of multi-polymerized MSP. |
| BBa_K5301007 | SnTST- mCh[11] | It is the third component of multi-polymerized MSP. |
Composite Parts
| Number | Name | Description | Basic Parts |
|---|---|---|---|
| BBa_K5301026 | sGFP1-10 tether expression pathway (T7) | This pathway expresses sGFP1-10 tether using T7 system. | [BBa_K2406020]-[BBa_B0012]-[BBa_K4623001]-[BBa_K5301017] |
| BBa_K5301027 | sGFP11 tether expression pathway (T7) | This pathway expresses sGFP11 tether using T7 system. | [BBa_K2406020]-[BBa_B0012]-[BBa_K4623001]-[BBa_K5301018] |
| BBa_K5301028 | spMSP1D1 expression pathway (T7) | This pathway expresses the SpyCatcher-MSP1D1-SpyTag fusion protein under IPTG-induced conditions, achieving self-cyclization of MSP1D1 to construct nanodiscs. | [BBa_K2406020]-[BBa_K731721]-[BBa_K5301011] |
| BBa_K5301019 | MSP1E3D1 expression pathway (T7) | This pathway expresses MSP1E3D1 using T7 expression system. | [BBa_K3633015]- [BBa_K3286004]- [BBa_K5301000]- [BBa_K731721] |
| BBa_K5301029 | spNW15 expression pathway (T7) | This pathway expresses the SpyCatcher-NW15-SpyTag fusion protein under IPTG induction, achieving self-cyclization of NW15 to construct nanodiscs. | [BBa_K2406020]- [BBa_K731721]- [BBa_K5301013] |
| BBa_K5301022 | spNW50 expression pathway (T7) | This pathway expresses the SpyCatcher-NW50-SpyTag fusion protein under IPTG induction, achieving self-cyclization of NW50 to construct nanodiscs. | [BBa_K2406020]- [BBa_K731721]- [BBa_K5301015] |
| BBa_K5301023 | SCSdC-mCh[1-10] expression pathway (T7) | This pathway expresses SCSdC-mCh[1-10] using T7 expression system. | [BBa_K3633015]- [BBa_K3286004]- [BBa_K5301005]- [BBa_K731721] |
| BBa_K5301020 | SnCSdT expression pathway (T7) | This pathway expresses SnCSdT using T7 expression system. | [BBa_K3633015]- [BBa_K3286004]- [BBa_K5301006]- [BBa_K731721] |
| BBa_K5301021 | SnTST-mCh[11] expression pathway (T7) | This pathway expresses SnTST-mCh[11] using T7 expression system. | [BBa_K3633015]- [BBa_K3286004]- [BBa_K5301007]- [BBa_K731721] |
| BBa_K5301024 | Multi-polymerized MSP | Multi-polymerized MSP is a larger MSP formed by connecting three MSPs with different linkers, which is conducive to forming larger nanodiscs. | [BBa_K5301005]- [BBa_K5301006]- [BBa_K5301007] |
References
[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.
[4] Schachter, I., et al., Confinement in Nanodiscs Anisotropically Modifies Lipid Bilayer Elastic Properties. The Journal of Physical Chemistry B, 2020. 124(33): p. 7166-7175.
