Difference between revisions of "Part:BBa K4623009"
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===Usage and Biology===
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==Usage and Biology==
 
Twisted Silinker (TS) is an intelligent recombinant protein that efficiently connects to the surface of silicon dioxide while undergoing conformational changes in response to environmental stimuli.
 
Twisted Silinker (TS) is an intelligent recombinant protein that efficiently connects to the surface of silicon dioxide while undergoing conformational changes in response to environmental stimuli.
  
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The sequence in the FASTA file has a His tag added, allowing purification of TS protein using a nickel column. An upstream TrxA fusion tag (BBa_K3619001) is added to aid in protein folding and reduce the formation of inclusion bodies in the bacterial host. After protein expression, thrombin cleavage exposes the mSA (BBa K4623001) site, allowing the binding of biotinylated functional proteins. CBP and calmodulin undergo conformational changes in the presence of calcium ions, tightly folding together to achieve the desired conformation. The SBP sequence can bind to the silicon dioxide surface, facilitating the modification of functional proteins onto the surface.
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The sequence added a His-tag , allowing purification of Twisted Silinker protein using a nickel column. An upstream TrxA fusion tag (BBa_K3619001) is added to aid in protein folding and reduce the formation of inclusion bodies in the bacterial host. After protein expression. CBP (BBa_K3755007) and calmodulin (BBa_I757003) undergo conformational changes in the presence of calcium ions, tightly folding together to achieve the desired conformation. The SBP (BBa_K4623001) sequence can bind to the silicon dioxide surface, facilitating the modification of functional proteins onto the surface.
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We transferred the pET-Duet1 plasmid into our engineered strain BL21 (DE3) and performed small-scale expression to determine the production conditions for His-tagged Twisted Silinker. The purified Twisted Silinker was detected using SDS-PAGE and Western Blot, with a molecular weight of 53kDa. To improve the purification strategy, we have also developed corresponding hardware for protein purification using the binding affinity between SBP and silicon dioxide, significantly enhancing the efficiency of protein production and purification. For reference, the hardware details can be found at our wiki.
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We transferred the pET-DUT1 plasmid into our engineered strain BL21(DE3) and performed small-scale expression to determine the production conditions for His-tagged Twisted Silinker. The purified Twisted Silinker was detected using SDS-PAGE and Western Blot, with a molecular weight of 53 kDa. To improve the purification strategy, we have also developed corresponding hardware for protein purification using the binding affinity between SBP and silicon dioxide, significantly enhancing the efficiency of protein production and purification.
 
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4623009 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4623009 SequenceAndFeatures</partinfo>
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==Cultivation, Purification and SDS-PAGE==
 
==Cultivation, Purification and SDS-PAGE==
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    In order to visualize the protein function of Twisted Silinker, we replaced the linker portion with a GFP sequence in subsequent experiments. This way, when CBP and calmodulin successfully bind together, the GFP protein will be activated, emitting green fluorescence.
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  To ensure proper folding of mSA and minimize inclusion body formation, we modified the protein buffer by adding biotin. The binding of biotin to mSA can help facilitate proper folding of the Twisted Silinker protein, reducing the formation of inclusion bodies resulting from misfolding. As a result, we obtained soluble protein extract in the supernatant. The formulation of the buffer and experimental procedures can be found in (protocol) for reference.
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    The presence of mSA monomers can easily lead to the formation of inclusion bodies, increasing the difficulty of purification. To achieve efficient expression of our Twisted Silinker and reduce the formation of inclusion bodies, we screened the IPTG induction conditions. We tested five different IPTG concentrations: 0 mM, 0.1 mM, 0.25 mM, and 0.5 mM. The results showed that the optimal concentration for protein expression was 0.1 mM.
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We chose 0.1mM IPTG for induction, and the target protein concentration was in a high level, which was the effective induction concentration.
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    To ensure proper folding of mSA and minimize inclusion body formation, we modified the protein buffer by adding biotin. The binding of biotin to mSA can help facilitate proper folding of the Twisted Silinker protein, reducing the formation of inclusion bodies resulting from misfolding. As a result, we obtained soluble protein extract in the supernatant. The formulation of the buffer and experimental procedures can be found in **(protocol)** for reference.
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     <img src="https://static.igem.wiki/teams/4623/wiki/ts-part/ts-linker/ts-part-1.png " alt="Image Description">
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<figcaption>figure 1 |Twisted Silinker protein IPTG concentration gradient-induced SDS-PAGE analysis. IPTG concentrations were set at 0, 0.1, 0.25, and 0.5 mM under 16°C induction conditions for 16 hours. A protein ladder, Blue Plus V Protein Marker with a range of 10-190 kDa, was used for comparison. The lanes on the gel are labeled as follows: marker, bacterial cell pellet (0 mM, 0.1 mM, 0.25 mM, 0.5 mM), supernatant (0 mM, 0.1 mM, 0.25 mM, 0.5 mM), bacterial cell lysate (0 mM, 0.1 mM, 0.25 mM, 0.5 mM). The SDS-PAGE gel was stained with Coomassie Brilliant Blue and protein bands were subsequently analyzed.</figcaption>
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    <figcaption>Figure 1 | Twisted Silinker Protein Purification SDS-PAGE gel. Massive expression of Twisted Silinker protein was performed under 16°C, 0.1mM IPTG induction, followed by nickel column affinity chromatography purification. A Blue Plus V Protein Marker (10-190kDa) was used for size comparison. Lane 1 represent the 500mM imidazole elution and Lane 2 represented the 500mM imidazole elution. The gel was stained with Coomassie brilliant blue and subjected to protein gel analysis. </figcaption>
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===Purification of Twisted Silinker===
 
===Purification of Twisted Silinker===
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After successfully determining the expression conditions of Twisted Silinker protein, it is necessary to scale up the culture and proceed with purification. We induced expression with 0.1mM IPTG and allowed it to proceed at 16°C for 20 hours, resulting in a large amount of the target protein. In the process of constructing the expression vector, we incorporated a His-tag into the target protein and utilized a nickel column for affinity chromatography purification based on the specific binding of the His-tagged protein.
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As shown in Figure 2, the bands displayed successful elution of a significant amount of the target protein using 500mM imidazole solution.
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     <img src="https://static.igem.wiki/teams/4623/wiki/ts-part/ts-linker/ts-part-2.png " alt="Image Description">
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After successfully determining the expression conditions of Twisted Silinker protein, it is necessary to scale up the culture and proceed with purification. We induced expression with 1 mM IPTG and allowed it to proceed at 16°C for 20 hours, resulting in a large amount of the target protein. In the process of constructing the expression vector, we incorporated a His-tag into the target protein and utilized a nickel column for affinity chromatography purification based on the specific binding of the His-tagged protein.
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     <figcaption>Figure 2 | Twisted Silinker Protein Purification SDS-PAGE gel. Massive expression of Twisted Silinker protein was performed under 16°C, 0.1mM IPTG induction, followed by nickel column affinity chromatography purification. A Blue Plus V Protein Marker (10-190kDa) was used for size comparison. Lane 1 represent the 500mM imidazole elution and Lane 2 represented the 500mM imidazole elution. The gel was stained with Coomassie brilliant blue and subjected to protein gel analysis. </figcaption>
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As shown in Figure 2, the bands displayed successful elution of a significant amount of the target protein using a 200 mM imidazole solution.
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     <img src="https://static.igem.wiki/teams/4623/wiki/bs-part/ts-part/ts-part/ts-f2.png" alt="TS figure2">
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     <figcaption>figure 2 |SDS-PAGE gel image of Twisted Silinker protein purification. Twisted Silinker protein was expressed at a large scale under 16°C with 0.1 mM IPTG induction, followed by purification using a nickel column via affinity chromatography. A protein ladder, Blue Plus V Protein Marker with a range of 10-190 kDa, was used as a reference. Lanes 1-5 in the image represent the elution fractions of Twisted Silinker protein using different imidazole concentrations: wash flow-through, 10 mM imidazole elution, 40 mM imidazole elution, 100 mM imidazole elution, and 200 mM imidazole elution, respectively. The gel was stained with Coomassie Brilliant Blue, and subsequent protein analysis was conducted.</figcaption>
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Latest revision as of 16:11, 11 October 2023


Twisted Silinker,Silica-protein junctions that curving in response to calcium ions


Usage and Biology

Twisted Silinker (TS) is an intelligent recombinant protein that efficiently connects to the surface of silicon dioxide while undergoing conformational changes in response to environmental stimuli.

The sequence added a His-tag , allowing purification of Twisted Silinker protein using a nickel column. An upstream TrxA fusion tag (BBa_K3619001) is added to aid in protein folding and reduce the formation of inclusion bodies in the bacterial host. After protein expression. CBP (BBa_K3755007) and calmodulin (BBa_I757003) undergo conformational changes in the presence of calcium ions, tightly folding together to achieve the desired conformation. The SBP (BBa_K4623001) sequence can bind to the silicon dioxide surface, facilitating the modification of functional proteins onto the surface.

We transferred the pET-Duet1 plasmid into our engineered strain BL21 (DE3) and performed small-scale expression to determine the production conditions for His-tagged Twisted Silinker. The purified Twisted Silinker was detected using SDS-PAGE and Western Blot, with a molecular weight of 53kDa. To improve the purification strategy, we have also developed corresponding hardware for protein purification using the binding affinity between SBP and silicon dioxide, significantly enhancing the efficiency of protein production and purification. For reference, the hardware details can be found at our wiki.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 955
    Illegal EcoRI site found at 1123
    Illegal EcoRI site found at 1345
    Illegal PstI site found at 1066
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 955
    Illegal EcoRI site found at 1123
    Illegal EcoRI site found at 1345
    Illegal PstI site found at 1066
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 955
    Illegal EcoRI site found at 1123
    Illegal EcoRI site found at 1345
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 955
    Illegal EcoRI site found at 1123
    Illegal EcoRI site found at 1345
    Illegal PstI site found at 1066
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 955
    Illegal EcoRI site found at 1123
    Illegal EcoRI site found at 1345
    Illegal PstI site found at 1066
    Illegal AgeI site found at 445
    Illegal AgeI site found at 505
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 940

Cultivation, Purification and SDS-PAGE

induction condition

To ensure proper folding of mSA and minimize inclusion body formation, we modified the protein buffer by adding biotin. The binding of biotin to mSA can help facilitate proper folding of the Twisted Silinker protein, reducing the formation of inclusion bodies resulting from misfolding. As a result, we obtained soluble protein extract in the supernatant. The formulation of the buffer and experimental procedures can be found in (protocol) for reference. We chose 0.1mM IPTG for induction, and the target protein concentration was in a high level, which was the effective induction concentration.

Image Description
Figure 1 | Twisted Silinker Protein Purification SDS-PAGE gel. Massive expression of Twisted Silinker protein was performed under 16°C, 0.1mM IPTG induction, followed by nickel column affinity chromatography purification. A Blue Plus V Protein Marker (10-190kDa) was used for size comparison. Lane 1 represent the 500mM imidazole elution and Lane 2 represented the 500mM imidazole elution. The gel was stained with Coomassie brilliant blue and subjected to protein gel analysis.

Purification of Twisted Silinker

After successfully determining the expression conditions of Twisted Silinker protein, it is necessary to scale up the culture and proceed with purification. We induced expression with 0.1mM IPTG and allowed it to proceed at 16°C for 20 hours, resulting in a large amount of the target protein. In the process of constructing the expression vector, we incorporated a His-tag into the target protein and utilized a nickel column for affinity chromatography purification based on the specific binding of the His-tagged protein. As shown in Figure 2, the bands displayed successful elution of a significant amount of the target protein using 500mM imidazole solution.


Image Description
Figure 2 | Twisted Silinker Protein Purification SDS-PAGE gel. Massive expression of Twisted Silinker protein was performed under 16°C, 0.1mM IPTG induction, followed by nickel column affinity chromatography purification. A Blue Plus V Protein Marker (10-190kDa) was used for size comparison. Lane 1 represent the 500mM imidazole elution and Lane 2 represented the 500mM imidazole elution. The gel was stained with Coomassie brilliant blue and subjected to protein gel analysis.