Difference between revisions of "Part:BBa K5301006"

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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].
 
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.
 
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.
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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 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.
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==Cultivation, Purification and SDS-PAGE==
 
==Cultivation, Purification and SDS-PAGE==

Revision as of 08:02, 1 October 2024


SnCSdT 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]. 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.

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. </div></div></div></div>

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 1.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 2.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]