Difference between revisions of "Part:BBa K4623012"

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(→‎Usage and Biology)
 
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===Usage and Biology===
 
===Usage and Biology===
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The Pairing Silinker (PS) is a novel recombinant protein that efficiently binds to the surface of silica and undergoes dimerization in response to specific signals on the silica surface. The sequence in the FASTA format includes an added HIS tag, enabling purification of the PS protein using a nickel column. Upstream of the sequence, a TrxA** (part number)** fusion tag is added to aid protein folding and reduce the formation of inclusion bodies in bacterial cells. Following protein expression, cleavage by thrombin** (part number)** exposes the mSA** (part number)** site, allowing binding to a biotinylated functional protein. The SBP** (part number)** sequence can bind to the silica surface, facilitating the modification of functional proteins onto the silica surface.
+
The Pairing Silinker (PS) is a novel recombinant protein that efficiently binds to the surface of silica and undergoes dimerization in response to specific signals on the silica surface. The sequence in the FASTA format includes an added HIS tag, enabling purification of the PS protein using a nickel column. Upstream of the sequence, a TrxA (BBa_K3619001) fusion tag is added to aid protein folding and reduce the formation of inclusion bodies in bacterial cells. Following protein expression, cleavage by thrombin exposes the mSA (BBa_K4623001) site, allowing binding to a biotinylated functional protein. The SBP (BBa_K4623000) sequence can bind to the silica surface, facilitating the modification of functional proteins onto the silica surface.
  
−
After introducing the pETDuet1 plasmid into our engineered BL21(DE3) bacteria, we conducted a small-scale trial expression and found that BL21(DE3) exhibited high basal expression levels, leading to the formation of PS inclusion bodies in uninduced strains. Therefore, in subsequent experiments, we employed the BL21(DE3) pLysS strain, which carries the pLysS plasmid and expresses T7 lysozyme to suppress leaky expression caused by T7 RNA polymerase. Experimental results demonstrated a significant increase in the solubility of PS.
+
 
 +
After introducing the pETDuet1 plasmid into our engineered <i>Escherichia coli</i> BL21(DE3) bacteria, we conducted a small-scale trial expression and found that <i>Escherichia coli</i> BL21(DE3) exhibited high basal expression levels, leading to the formation of PS inclusion bodies in uninduced strains. Therefore, in subsequent experiments, we employed the <i>Escherichia coli</i> BL21(DE3) pLysS strain, which carries the pLysS plasmid and expresses T7 lysozyme to suppress leaky expression caused by T7 RNA polymerase. Experimental results demonstrated a significant increase in the solubility of PS.
  
 
Purified Pairing Silinker can be detected via SDS-PAGE, with a molecular weight of approximately 44 kDa, higher than the theoretical value of 34 kDa. This discrepancy may be attributed to flexible C-terminal sequences like SBP. To enhance the purification strategy, we have also developed corresponding hardware, utilizing the binding affinity between SBP and silica to purify the protein. This greatly improves the efficiency of protein production and purification, reducing costs.
 
Purified Pairing Silinker can be detected via SDS-PAGE, with a molecular weight of approximately 44 kDa, higher than the theoretical value of 34 kDa. This discrepancy may be attributed to flexible C-terminal sequences like SBP. To enhance the purification strategy, we have also developed corresponding hardware, utilizing the binding affinity between SBP and silica to purify the protein. This greatly improves the efficiency of protein production and purification, reducing costs.
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The presence of mSA monomers indeed tends to promote protein aggregation and inclusion body formation, making purification more challenging.
 
The presence of mSA monomers indeed tends to promote protein aggregation and inclusion body formation, making purification more challenging.
  
−
To achieve efficient expression of PS while reducing inclusion body formation, we conducted tests on the culture conditions of our BL21(DE3) engineered bacteria. We set up four different culture media: Media A: LB + 50 mM KH2PO4, 50 mM Na2HPO4, 25 mM (NH4)2SO4, and 2 mM MgSO4; A + 50 mM mannitol; A + 2% ethanol; A + 4% glycerol. Experimental results showed that the addition of media additives did not significantly reduce inclusion body formation or improve protein solubility expression. Furthermore, the control group indicated another issue: BL21(DE3) exhibited excessive leaky expression, as evident from the presence of prominent inclusion bodies even in uninduced bacterial cells.
+
To achieve efficient expression of PS while reducing inclusion body formation, we conducted tests on the culture conditions of our <i>E.coli</i> BL21(DE3) engineered bacteria. We set up four different culture media: Media A: LB + 50 mM KH<sub>2</sub>PO<sub>4</sub>, 50 mM Na<sub>2</sub>HPO<sub>4</sub>, 25 mM (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, and 2 mM MgSO<sub>4</sub>; A + 50 mM mannitol; A + 2% ethanol; A + 4% glycerol. Experimental results showed that the addition of media additives did not significantly reduce inclusion body formation or improve protein solubility expression. Furthermore, the control group indicated another issue: <i>E.coli</i> BL21(DE3) exhibited excessive leaky expression, as evident from the presence of prominent inclusion bodies even in uninduced bacterial cells.
 
      
 
      
 
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    <img src="https://static.igem.wiki/teams/4623/wiki/ps-part/ps-part/ps-peiyangji-tiaojian.png" alt="ps f1">
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    <figcaption>Figure 1 | SDS-PAGE analysis of Pairing Silinker protein expression induced with different media showed IPTG concentration gradients of 0.1mM for 16 h at 16 ° C.  A 10-190kDa Blue Plus V Protein Marker was used for protein size comparison. The lane bands in the image represent the marker; 1：soluble fraction after cell lysis; 2: insoluble fraction after cell lysis; Contrl：insoluble fraction from uninduced cell; medium A: 50 mM KH<sub>2</sub>PO<sub>4</sub>, 50mM Na<sub>2</sub>HPO<sub>4</sub>,   25mM (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, and 2mM MgSO<sub>4</sub>; medium B: medium A with 50mM sorbitol; medium C: medium A with 2% alcohol; medium D: medium A with 4% glycerine. The gel was run at 80V for 10 min and then at 150V for 20 min, followed by staining with Coomassie Brilliant blue dye. “ * ”Marked as the target band size</figcaption>
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Therefore, we switched to the expression strain <i>E.coli</i>  BL21(DE3) pLysS, which carries the <i>E.coli</i> BL21 (DE3) pLysS plasmid and expresses T7 lysozyme, effectively inhibiting basal expression. Experimental results confirmed that <i>E.coli</i> BL21(DE3) pLysS exhibited significantly higher soluble expression compared to <i>E.coli</i> BL21(DE3).
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    <img src="https://static.igem.wiki/teams/4623/wiki/ps-part/ps-part/ps-soluble-expression.png" alt="ps f2">
 +
    <figcaption>Figure 2 | SDS-PAGE analysis of Pairing Silinker protein expression in <i>E.coli</i> BL21 (DE3) pLysS, induced at 16°C for 16 hours. Protein sizes were compared using a Blue Plus V protein marker ranging from 10-190 kDa. Lanes in the figure, from left to right, represent: 1-3: Cell samples; 4-6: Soluble fraction after cell lysis; 7-9: Insoluble fraction after cell lysis. The gel was run at 80V for 10 minutes, followed by 150V for 20 minutes, and then stained with Coomassie Brilliant Blue dye. </figcaption>
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  <p></p >
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</body>
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</html>
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To ensure proper folding of mSA and reduce inclusion body formation, the protein's buffer must contain biotin. As shown in the experimental results, in the absence of biotin, the protein primarily existed in precipitated form. Figure 3 shows the results of SDS-PAGE analysis of protein-induced culture products without the addition of biotin to the protein buffer.
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<body>
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    <img src="https://static.igem.wiki/teams/4623/wiki/ps-part/ps-part/ps-plyss1.png " alt="ps f3">
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    <figcaption> Figure 3 | SDS-PAGE analysis of Pairing Silinker protein expression in <i>E.coli</i> BL21 (DE3) pLysS  at 16°C for 16 hours. Protein sizes were compared using a Blue Plus V protein marker ranging from 10-190 kDa. Lanes in the figure, from left to right, represent: 1-2: Cell samples; 3-4: insoluble fraction after cell lysis; 5-6: soluble fraction after cell lysis. The gel was run at 80V for 10 minutes, followed by 150V for 20 minutes, and then stained with Coomassie Brilliant Blue dye.</figcaption>
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  <p></p >
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</body>
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===Purification of Basic Silinker===
 
===Purification of Basic Silinker===
−
===Functional examination of PS proteins===
+
After successfully obtaining the PS protein, it is necessary to scale up the cultivation and perform purification of the target protein. We induced large-scale expression using 0.1 mM IPTG at 16°C for 16-20 hours to produce a significant amount of the target protein. His-tag affinity chromatography was utilized for purification. As shown in the following figure, a substantial amount of the target protein was successfully eluted using 500 mM imidazole, resulting in prominent bands.
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    <img src="https://static.igem.wiki/teams/4623/wiki/ps-part/ps-part/ps-ni-column.png " ps f5">
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    <figcaption>Figure 4 | SDS-PAGE analysis of PS protein purification. Large-scale expression of PS was inducted at 16°C with 0.1mM IPTG induction, followed by purification using nickel affinity chromatography. A 10-170kDa BeyoColor protein marker was used for size comparison. From lanes 1 to 6, they represent : 1:supernatants after cells lysis,2:flow-through, 3-5:10mM imidazole elution , 6.500mM imidazole elution .The gel was run at 80V for 10 minutes and then at 150V for 20 minutes, followed by staining with Coomassie Brilliant Blue dye. </figcaption>
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  </figure>
 +
  <p></p >
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</body>
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</html>
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==Functional examination of PS proteins==
 
==Functional examination of PS proteins==
 +
We verified the binding ability of PS to silica, and as shown in Figure 5, PS can bind silica well. Based on the effect of tween-20 on the ability of silinker to bind silica given by BS experiments, we tested the effect of adding tween-20 alone.
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<body>
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    <img src="https://static.igem.wiki/teams/4623/wiki/ps-part/ps-part/ps-sio2-binding.png" alt="ps f6">
 +
    <figcaption>Figure 5 | Binding results of PS protein and silica. Protein sizes were compared using a Blue Plus V protein marker ranging from 10-190 kDa. Lanes in the figure, from left to right, represent: PS: PS protein solution, FT: flow through, W1-3: washing by PBS, 1: W3 sample eluted with PBS+2% tween-20, 2: Sample from 1 with added protein loading buffer and boiling, 3: Sample from W3 with added protein loading buffer and boiling. The gel was run at 80V for 10 minutes, followed by 150V for 20 minutes, and then stained with Coomassie Brilliant Blue dye. </figcaption>
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  <p></p >
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</body>
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</html>
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FIG. 5 Results prove that PS protein can bind silica and is difficult to elute, and tween-20 cannot elute PS protein when used alone.We observed a prominent band at approximately 90 kDa, which was not present in the initially purified PS protein (Figure 4). We speculate that this is indicative of PS dimer formation (with monomers approximately 44 kDa each), aligning with our design expectations for PS. Therefore, we aimed to further validate the dimerization of PS in the presence of calcium ions. However, our attempts to separate dimers using size exclusion chromatography and obtain dimer bands using Native-PAGE yielded puzzling results. In the size exclusion experiment, we did not observe any UV absorption peak in the chromatogram, and after staining the Native-PAGE gel, there were no bands. This is a frustrating outcome, and we suspect it may be due to the inevitable presence of silica in the instruments used for both size exclusion chromatography and Native-PAGE.
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K4623012 parameters</partinfo>
 
<partinfo>BBa_K4623012 parameters</partinfo>
 
<!-- -->
 
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Latest revision as of 13:54, 11 October 2023


Pairing Silinker, dimerization in response to specific signals on the silica surface

Usage and Biology

The Pairing Silinker (PS) is a novel recombinant protein that efficiently binds to the surface of silica and undergoes dimerization in response to specific signals on the silica surface. The sequence in the FASTA format includes an added HIS tag, enabling purification of the PS protein using a nickel column. Upstream of the sequence, a TrxA (BBa_K3619001) fusion tag is added to aid protein folding and reduce the formation of inclusion bodies in bacterial cells. Following protein expression, cleavage by thrombin exposes the mSA (BBa_K4623001) site, allowing binding to a biotinylated functional protein. The SBP (BBa_K4623000) sequence can bind to the silica surface, facilitating the modification of functional proteins onto the silica surface.


After introducing the pETDuet1 plasmid into our engineered Escherichia coli BL21(DE3) bacteria, we conducted a small-scale trial expression and found that Escherichia coli BL21(DE3) exhibited high basal expression levels, leading to the formation of PS inclusion bodies in uninduced strains. Therefore, in subsequent experiments, we employed the Escherichia coli BL21(DE3) pLysS strain, which carries the pLysS plasmid and expresses T7 lysozyme to suppress leaky expression caused by T7 RNA polymerase. Experimental results demonstrated a significant increase in the solubility of PS.

Purified Pairing Silinker can be detected via SDS-PAGE, with a molecular weight of approximately 44 kDa, higher than the theoretical value of 34 kDa. This discrepancy may be attributed to flexible C-terminal sequences like SBP. To enhance the purification strategy, we have also developed corresponding hardware, utilizing the binding affinity between SBP and silica to purify the protein. This greatly improves the efficiency of protein production and purification, reducing costs.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 445
    Illegal AgeI site found at 505
  • 1000
    COMPATIBLE WITH RFC[1000]

Cultivation, Purification and SDS-PAGE

induction condition

The presence of mSA monomers indeed tends to promote protein aggregation and inclusion body formation, making purification more challenging. To achieve efficient expression of PS while reducing inclusion body formation, we conducted tests on the culture conditions of our E.coli BL21(DE3) engineered bacteria. We set up four different culture media: Media A: LB + 50 mM KH2PO4, 50 mM Na2HPO4, 25 mM (NH4)2SO4, and 2 mM MgSO4; A + 50 mM mannitol; A + 2% ethanol; A + 4% glycerol. Experimental results showed that the addition of media additives did not significantly reduce inclusion body formation or improve protein solubility expression. Furthermore, the control group indicated another issue: E.coli BL21(DE3) exhibited excessive leaky expression, as evident from the presence of prominent inclusion bodies even in uninduced bacterial cells.

ps f1
Figure 1 | SDS-PAGE analysis of Pairing Silinker protein expression induced with different media showed IPTG concentration gradients of 0.1mM for 16 h at 16 ° C. A 10-190kDa Blue Plus V Protein Marker was used for protein size comparison. The lane bands in the image represent the marker; 1：soluble fraction after cell lysis; 2: insoluble fraction after cell lysis; Contrl：insoluble fraction from uninduced cell; medium A: 50 mM KH2PO4, 50mM Na2HPO4,   25mM (NH4)2SO4, and 2mM MgSO4; medium B: medium A with 50mM sorbitol; medium C: medium A with 2% alcohol; medium D: medium A with 4% glycerine. The gel was run at 80V for 10 min and then at 150V for 20 min, followed by staining with Coomassie Brilliant blue dye. “ * ”Marked as the target band size


Therefore, we switched to the expression strain E.coli BL21(DE3) pLysS, which carries the E.coli BL21 (DE3) pLysS plasmid and expresses T7 lysozyme, effectively inhibiting basal expression. Experimental results confirmed that E.coli BL21(DE3) pLysS exhibited significantly higher soluble expression compared to E.coli BL21(DE3).


ps f2
Figure 2 | SDS-PAGE analysis of Pairing Silinker protein expression in E.coli BL21 (DE3) pLysS, induced at 16°C for 16 hours. Protein sizes were compared using a Blue Plus V protein marker ranging from 10-190 kDa. Lanes in the figure, from left to right, represent: 1-3: Cell samples; 4-6: Soluble fraction after cell lysis; 7-9: Insoluble fraction after cell lysis. The gel was run at 80V for 10 minutes, followed by 150V for 20 minutes, and then stained with Coomassie Brilliant Blue dye.

To ensure proper folding of mSA and reduce inclusion body formation, the protein's buffer must contain biotin. As shown in the experimental results, in the absence of biotin, the protein primarily existed in precipitated form. Figure 3 shows the results of SDS-PAGE analysis of protein-induced culture products without the addition of biotin to the protein buffer.


ps f3
Figure 3 | SDS-PAGE analysis of Pairing Silinker protein expression in E.coli BL21 (DE3) pLysS at 16°C for 16 hours. Protein sizes were compared using a Blue Plus V protein marker ranging from 10-190 kDa. Lanes in the figure, from left to right, represent: 1-2: Cell samples; 3-4: insoluble fraction after cell lysis; 5-6: soluble fraction after cell lysis. The gel was run at 80V for 10 minutes, followed by 150V for 20 minutes, and then stained with Coomassie Brilliant Blue dye.

Purification of Basic Silinker

After successfully obtaining the PS protein, it is necessary to scale up the cultivation and perform purification of the target protein. We induced large-scale expression using 0.1 mM IPTG at 16°C for 16-20 hours to produce a significant amount of the target protein. His-tag affinity chromatography was utilized for purification. As shown in the following figure, a substantial amount of the target protein was successfully eluted using 500 mM imidazole, resulting in prominent bands.

Figure 4 | SDS-PAGE analysis of PS protein purification. Large-scale expression of PS was inducted at 16°C with 0.1mM IPTG induction, followed by purification using nickel affinity chromatography. A 10-170kDa BeyoColor protein marker was used for size comparison. From lanes 1 to 6, they represent : 1:supernatants after cells lysis,2:flow-through, 3-5:10mM imidazole elution , 6.500mM imidazole elution .The gel was run at 80V for 10 minutes and then at 150V for 20 minutes, followed by staining with Coomassie Brilliant Blue dye.

Functional examination of PS proteins

We verified the binding ability of PS to silica, and as shown in Figure 5, PS can bind silica well. Based on the effect of tween-20 on the ability of silinker to bind silica given by BS experiments, we tested the effect of adding tween-20 alone.

ps f6
Figure 5 | Binding results of PS protein and silica. Protein sizes were compared using a Blue Plus V protein marker ranging from 10-190 kDa. Lanes in the figure, from left to right, represent: PS: PS protein solution, FT: flow through, W1-3: washing by PBS, 1: W3 sample eluted with PBS+2% tween-20, 2: Sample from 1 with added protein loading buffer and boiling, 3: Sample from W3 with added protein loading buffer and boiling. The gel was run at 80V for 10 minutes, followed by 150V for 20 minutes, and then stained with Coomassie Brilliant Blue dye.

FIG. 5 Results prove that PS protein can bind silica and is difficult to elute, and tween-20 cannot elute PS protein when used alone.We observed a prominent band at approximately 90 kDa, which was not present in the initially purified PS protein (Figure 4). We speculate that this is indicative of PS dimer formation (with monomers approximately 44 kDa each), aligning with our design expectations for PS. Therefore, we aimed to further validate the dimerization of PS in the presence of calcium ions. However, our attempts to separate dimers using size exclusion chromatography and obtain dimer bands using Native-PAGE yielded puzzling results. In the size exclusion experiment, we did not observe any UV absorption peak in the chromatogram, and after staining the Native-PAGE gel, there were no bands. This is a frustrating outcome, and we suspect it may be due to the inevitable presence of silica in the instruments used for both size exclusion chromatography and Native-PAGE.