Difference between revisions of "Part:BBa K5301007"

Revision as of 08:08, 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.

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 SnoopTag and SpyTag at its N-terminus and C-terminus to form SnTST-mCh[11]. 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.

Figure 1.Colony PCR Results of Host Bacteria Introduced with pET28a_SnTST-mCh[11], All Four Colonies Successfully Obtained the Desired Band, Indicating Successful Plasmid Transformation.The theoretical sequence length is 911 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. To achieve efficient expression of SnTST-mCh[11], we screened the induction conditions using IPTG. We set up a gradient of four IPTG concentrations: 0.2mM, 0.5mM, 0.8mM, and 1mM. The results showed that the optimal protein expression was achieved with a concentration of 0.8mM.

Figure 1.SDS-PAGE analysis of SnTST-mCh[11] protein with IPTG concentration gradient induction. An IPTG concentration gradient of 0.2mM, 0.5mM, 0.8mM, and 1mM was used for 16 hours induction at 16°C. The molecular weight of SnTST-mCh[11] is 33.6 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 100mM and 500mM imidazole, and successfully obtained target protein. To verify the successful expression of the protein, we conducted a Western Blot using anti-Histag after SDS-PAGE and confirmed that we had obtained SnTST-mCh[11] with a relatively high purity.

Figure 2.Western Blot Analysis of SCSdC-mCh[1-10] Purified by Nickel Affinity Chromatography. The presence of the target protein in the 100mM imidazole elution buffer was verified through Histag antibody and the correct molecular weight of the bands. The molecular weight of SnTST-mCh[11] is 33.6 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 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]

@@ Line 7: / Line 7: @@
 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.
+==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 SnoopTag and SpyTag at its N-terminus and C-terminus to form SnTST-mCh[11]. 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.
+<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:min-content;"><div style="zoom:0.5;overflow:hidden;">
+https://static.igem.wiki/teams/5301/parts/tri-3-pcr.png
+</div><div class="thumbcaption">
+Figure 1.Colony PCR Results of Host Bacteria Introduced with pET28a_SnTST-mCh[11], All Four Colonies Successfully Obtained the Desired Band, Indicating Successful Plasmid Transformation.The theoretical sequence length is 911 bp.
+</div></div></div></div>
 ==Cultivation, Purification and SDS-PAGE==