Difference between revisions of "Part:BBa K5301007"
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<partinfo>BBa_K5301007 short</partinfo>
 
<partinfo>BBa_K5301007 short</partinfo>
  
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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].
 
SnTST-mCh[11], the third component of multi-polymerized MSP, is a fusion protein composed of SnoopTag, NW15, mCherry [11], and SpyTag, with flexible GS linkers used to connect each part.
 
SnTST-mCh[11], the third component of multi-polymerized MSP, is a fusion protein composed of SnoopTag, NW15, mCherry [11], and SpyTag, with flexible GS linkers used to connect each part.
  
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==Cultivation, Purification and SDS-PAGE==
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===Usage and Biology===
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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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<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:min-content;"><div style="zoom:0.5;overflow:hidden;">
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https://static.igem.wiki/teams/5301/parts/tri-1-induction.png
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Figure 1.SDS-PAGE analysis of SnTST-mCh[11] protein with IPTG concentration gradient induction. An IPTG concentration of 0.8mM was used for 16 hours induction at 16°C. The molecular weight of SnTST-mCh[11] is 76.3 kDa.
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===Purification===
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After confirming the successful expression of SnTST-mCh[11], 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.
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<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:min-content;"><div style="zoom:0.4;overflow:hidden;">
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https://static.igem.wiki/teams/5301/parts/tri-1-purification.png
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</div><div class="thumbcaption">
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Figure 2.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 SnTST-mCh[11] is 76.3 kDa.
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Revision as of 04:04, 1 October 2024


SnTST-mCh[11] is one of the components of multi-polymerized MSP.

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]. SnTST-mCh[11], the third component of multi-polymerized MSP, is a fusion protein composed of SnoopTag, NW15, mCherry [11], and SpyTag, with flexible GS linkers used to connect each part.

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 1.SDS-PAGE analysis of SnTST-mCh[11] protein with IPTG concentration gradient induction. An IPTG concentration of 0.8mM was used for 16 hours induction at 16°C. The molecular weight of SnTST-mCh[11] is 76.3 kDa.

Purification

After confirming the successful expression of SnTST-mCh[11], 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-1-purification.png

Figure 2.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 SnTST-mCh[11] is 76.3 kDa.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 757
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 276
    Illegal BglII site found at 648
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]