Difference between revisions of "Part:BBa K5301006"

Revision as of 03:34, 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.

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.

Figure 1.SDS-PAGE analysis of SnCSdT 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 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.

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

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]

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 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.
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-https://static.igem.wiki/teams/5301/parts/tri-1-purification.png
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 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.