Difference between revisions of "Team:Vilnius-Lithuania/Design"

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<p>In order to reconstitute the fully working Bam complex we relied on the mechanisms elucidated before. SurA is a periplasmic chaperone which binds to unfolded β-barrel proteins and retains them in unfolded state, thus preventing aggregation. BamB or BamD lipoproteins can bind BamA-SurA, direct the complex to the membrane, and catalyze BamA insertion as well as correct folding in the membrane. [9] (Fig. 3, step 1) Knowing that in bacteria most of the Bam lipoproteins are found in BAM complex and full five proteins complex can be purified with one tag without any cross-links, we expected high complex association constants among the components (Fig.3, step 2) and hypothesized that BAM complex could be assembled in vitro simply by encapsulating BamA-SurA associatives and Bam lipoproteins.</p>
 
<p>In order to reconstitute the fully working Bam complex we relied on the mechanisms elucidated before. SurA is a periplasmic chaperone which binds to unfolded β-barrel proteins and retains them in unfolded state, thus preventing aggregation. BamB or BamD lipoproteins can bind BamA-SurA, direct the complex to the membrane, and catalyze BamA insertion as well as correct folding in the membrane. [9] (Fig. 3, step 1) Knowing that in bacteria most of the Bam lipoproteins are found in BAM complex and full five proteins complex can be purified with one tag without any cross-links, we expected high complex association constants among the components (Fig.3, step 2) and hypothesized that BAM complex could be assembled in vitro simply by encapsulating BamA-SurA associatives and Bam lipoproteins.</p>
 
<div class="image-container">
 
<div class="image-container">
     <img src="https://static.igem.org/mediawiki/2018/3/3a/T--Vilnius-Lithuania--Fig2_BAM_compl.png"/>
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     <img src="https://static.igem.org/mediawiki/2018/3/30/T--Vilnius-Lithuania--Fig3_BAM_compl.png"/>
 
<p><strong>Fig. 3</strong> Bam lipoproteins assemble BamA in vitro</p>
 
<p><strong>Fig. 3</strong> Bam lipoproteins assemble BamA in vitro</p>
 
<p></p>
 
<p></p>
 
<h1>Results</h1>
 
<h1>Results</h1>
<h2>Plasmid construction</h2>>
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<h2>Plasmid construction</h2>
 
<p>To purify the components of BAM complex we have constructed 6 plasmids:</p>
 
<p>To purify the components of BAM complex we have constructed 6 plasmids:</p>
 
<p>
 
<p>
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<p>
 
<p>
 
     <div class="image-container">
 
     <div class="image-container">
        <img src="https://static.igem.org/mediawiki/2018/3/30/T--Vilnius-Lithuania--Fig3_BAM_compl.png"/><div class="image-container">
 
 
             <img src="https://static.igem.org/mediawiki/2018/5/54/T--Vilnius-Lithuania--Fig4.1_BAM_compl.png"/><div class="image-container">
 
             <img src="https://static.igem.org/mediawiki/2018/5/54/T--Vilnius-Lithuania--Fig4.1_BAM_compl.png"/><div class="image-container">
                 <img src="https://static.igem.org/mediawiki/2018/3/3d/T--Vilnius-Lithuania--Fig4.2_BAM_compl.png"/><div class="image-container">
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                 <img src="https://static.igem.org/mediawiki/2018/3/3d/T--Vilnius-Lithuania--Fig4.2_BAM_compl.png"/><div class="image-container"></p>
                    <img src="https://static.igem.org/mediawiki/2018/2/2e/T--Vilnius-Lithuania--Fig5_BAM_compl.png"/><div class="image-container">
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                        <img src="https://static.igem.org/mediawiki/2018/8/85/T--Vilnius-Lithuania--Fig6_BAM_compl.png"/><div class="image-container">
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                            <img src="https://static.igem.org/mediawiki/2018/2/2c/T--Vilnius-Lithuania--Fig7_BAM_compl.png"/>
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</p>
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<p><strong>Fig. 4</strong> Maps of constructed plasmids</p>
 
<p><strong>Fig. 4</strong> Maps of constructed plasmids</p>
 
div class="image-container">
 
 
         <img src="https://static.igem.org/mediawiki/2018/2/2e/T--Vilnius-Lithuania--Fig5_BAM_compl.png"/><div class="image-container">
 
         <img src="https://static.igem.org/mediawiki/2018/2/2e/T--Vilnius-Lithuania--Fig5_BAM_compl.png"/><div class="image-container">
 
<p><strong>Fig. 5</strong> PCR products of genes of BAM complex 1,18 – GeneRuler 1 kb DNA ladder;  4,5 – SurA (1250bp); 6,7 -  BamE (390bp); 8,9 – BamB (1201bp); 10, 11 – BamC (1056bp); 12, 13 – BamD(756bp); 14,16 – BamA (2455bp)</p>
 
<p><strong>Fig. 5</strong> PCR products of genes of BAM complex 1,18 – GeneRuler 1 kb DNA ladder;  4,5 – SurA (1250bp); 6,7 -  BamE (390bp); 8,9 – BamB (1201bp); 10, 11 – BamC (1056bp); 12, 13 – BamD(756bp); 14,16 – BamA (2455bp)</p>
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     <li>For our experiments we needed an unfolded BamA. Therefore, we overexpressed BamA , which we isolated in the form of inclusion bodies and then dissolved in 8M urea without any further purification steps.</li>
 
     <li>For our experiments we needed an unfolded BamA. Therefore, we overexpressed BamA , which we isolated in the form of inclusion bodies and then dissolved in 8M urea without any further purification steps.</li>
 
</ul>
 
</ul>
https://static.igem.org/mediawiki/2018/8/85/T--Vilnius-Lithuania--Fig6_BAM_compl.png
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<div class="image-container">
<strong>Fig. 6</strong> BamA after purification
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    <img src="https://static.igem.org/mediawiki/2018/8/85/T--Vilnius-Lithuania--Fig6_BAM_compl.png"/>
1 – BamA, 2 – BamA-HisN6, L – PageRuler Unstained Broad Range Protein Ladder
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<p><strong>Fig. 6</strong> BamA after purification
<ul>
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    1 – BamA, 2 – BamA-HisN6, L – PageRuler Unstained Broad Range Protein Ladder</p>
 +
    <ul>
 
<Li>Bam B-D lipoproteins were expressed with the pelB signal sequence, leading them to be exported to the periplasm where lipidation takes place. We then isolated the proteins from the membrane fraction, which we solubilised with specific detergents before purification using Ni-NTA (Fig.7 and Fig.9) and gelfiltration (size-exclusion) (Fig. 8 and Fig. 9) columns. BamCDE were purified as a single subcomplex via one octahistidine tag on BamE.</Li>
 
<Li>Bam B-D lipoproteins were expressed with the pelB signal sequence, leading them to be exported to the periplasm where lipidation takes place. We then isolated the proteins from the membrane fraction, which we solubilised with specific detergents before purification using Ni-NTA (Fig.7 and Fig.9) and gelfiltration (size-exclusion) (Fig. 8 and Fig. 9) columns. BamCDE were purified as a single subcomplex via one octahistidine tag on BamE.</Li>
 
</ul>
 
</ul>
 
<h3>After purification with Ni-NTA column:</h3>
 
<h3>After purification with Ni-NTA column:</h3>
 
<p>
 
<p>
     Fig. 7
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     <img src="https://static.igem.org/mediawiki/2018/2/2c/T--Vilnius-Lithuania--Fig7_BAM_compl.png"/>
 
</p>
 
</p>
<strong>Fig. 7</strong> BamB after Ni-NTA column purification Lane L – PageRuler Unstained Protein Ladder, Lane 1 – Sample  loaded on Ni-NTA Column,
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<p><strong>Fig. 7</strong> BamB after Ni-NTA column purification Lane L – PageRuler Unstained Protein Ladder, Lane 1 – Sample  loaded on Ni-NTA Column,
Lanes 2-3 – Flow through fractions, Lanes 4-5 – washing fractions, Lanes 6-9 – Elution fractions
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    Lanes 2-3 – Flow through fractions, Lanes 4-5 – washing fractions, Lanes 6-9 – Elution fractions
 
+
    </p>
 
<h3> After gelfiltration:</h3>
 
<h3> After gelfiltration:</h3>
  
 
<p>
 
<p>
     Fig. 8
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     <img src="https://static.igem.org/mediawiki/2018/2/2e/T--Vilnius-Lithuania--Fig8_BAM_compl.png"/>
 
</p>
 
</p>
<strong>Fig. 8</strong>BamB fractions after gelfiltration
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<p><strong>Fig. 8</strong>BamB fractions after gelfiltration
 
Lane L – PageRuler Unstained Protein Ladder, Lanes 1-14 Elution fractions
 
Lane L – PageRuler Unstained Protein Ladder, Lanes 1-14 Elution fractions
 
+
</p>
 
<h3> After gelfiltration:</h3>
 
<h3> After gelfiltration:</h3>
  
 
<p>
 
<p>
     Fig. 9
+
     <img src="https://static.igem.org/mediawiki/2018/3/36/T--Vilnius-Lithuania--Fig9_BAM_compl.png"/>
 +
</p>
 +
<p><strong>Fig. 9</strong> BamCDE after Ni-NTA column purification Lane L – PageRuler Unstained Protein Ladder, Lane 1,2 - Flow through fractions, Lanes 3-4 – washing fractions, Lanes 5-7 – Elution fractions
 
</p>
 
</p>
<strong>Fig. 9</strong> BamCDE after Ni-NTA column purification Lane L – PageRuler Unstained Protein Ladder, Lane 1,2 - Flow through fractions, Lanes 3-4 – washing fractions, Lanes 5-7 – Elution fractions
 
 
 
<h3>After gelfiltration:</h3>
 
<h3>After gelfiltration:</h3>
  
 
<p>
 
<p>
     Fig. 10
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     <img src="https://static.igem.org/mediawiki/2018/a/ae/T--Vilnius-Lithuania--Fig10_BAM_compl.png"/>
 
</p>
 
</p>
<strong>Fig. 10</strong> BamCDE fractions after gelfiltration
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<p><strong>Fig. 10</strong> BamCDE fractions after gelfiltration
Lane 1 – PageRuler Unstained Protein Ladder,  2-15 elution fractions
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    Lane 1 – PageRuler Unstained Protein Ladder,  2-15 elution fractions</p><ul>
<ul>
+
 
     <li>
 
     <li>
 
         While SurA is a periplasmic protein, we had no issues overexpressing it in the cytoplasm for increased yield. We used a hexahistidine tag and purified using Ni-NTA (Fig.11) and gelfiltration (Fig.12) columns.
 
         While SurA is a periplasmic protein, we had no issues overexpressing it in the cytoplasm for increased yield. We used a hexahistidine tag and purified using Ni-NTA (Fig.11) and gelfiltration (Fig.12) columns.
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</ul>
 
</ul>
 
<h3>After Ni-NTA column:</h3>
 
<h3>After Ni-NTA column:</h3>
<strong>Fig. 11 </strong>SurA after purification with Ni-NTA column
+
<img src="https://static.igem.org/mediawiki/2018/3/33/T--Vilnius-Lithuania--Fig11_BAM_compl.png"/>
L - PageRuler Unstained Protein Ladder, 1 - Protein loaded on Ni-NTA Column, Lane 2 – Flow through fraction, 3 - washing fraction, 4-9 elution fractions
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<p><strong>Fig. 11 </strong>SurA after purification with Ni-NTA column
 
+
    L - PageRuler Unstained Protein Ladder, 1 - Protein loaded on Ni-NTA Column, Lane 2 – Flow through fraction, 3 - washing fraction, 4-9 elution fractions
 +
    </p>
 
<h3>After gelfiltration</h3>
 
<h3>After gelfiltration</h3>
 
<p>
 
<p>
     Fig. 12
+
     <img src="https://static.igem.org/mediawiki/2018/e/e3/T--Vilnius-Lithuania--Fig12_BAM_compl.png"/>
 
</p>
 
</p>
<strong>Fig. 12 </strong>SurA fractions after gelfiltration
+
<p><strong>Fig. 12 </strong>SurA fractions after gelfiltration
L - PageRuler Unstained Protein Ladder, 1-9 elution fractions  
+
    L - PageRuler Unstained Protein Ladder, 1-9 elution fractions  
<H2>Folding assay</H2>
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    </p><H2>Folding assay</H2>
 
<p>To determine whether the purified proteins act as expected we conducted a specific a folding assay. Proteins possessing β-barrel structures exhibit a unique characteristic - when mixed with SDS (for SDS-PAGE) but unboiled, the β-barrel structure remains intact, which causes the protein to move differently in the SDS-PAGE gel in comparison to the same protein lacking these structures - which occurs when it is denatured or did not originally fold. Exploiting this characteristic makes it possible to observe and quantify protein folding levels.</p>
 
<p>To determine whether the purified proteins act as expected we conducted a specific a folding assay. Proteins possessing β-barrel structures exhibit a unique characteristic - when mixed with SDS (for SDS-PAGE) but unboiled, the β-barrel structure remains intact, which causes the protein to move differently in the SDS-PAGE gel in comparison to the same protein lacking these structures - which occurs when it is denatured or did not originally fold. Exploiting this characteristic makes it possible to observe and quantify protein folding levels.</p>
 
<p>For the first experiment we observed if BamB and BamCDE can incorporate the unfolded BamA protein into the membrane and reconstitute the complete BAM complex. This was accomplished by incubating SurA with BamA denatured in urea, then transferring it into a solution featuring liposomes, BamB and BamCDE,  then further incubating for 2 hours. As BamA was expressed with a his-tag, we performed a blot to determine the level of protein folding (Fig. 13). </p>
 
<p>For the first experiment we observed if BamB and BamCDE can incorporate the unfolded BamA protein into the membrane and reconstitute the complete BAM complex. This was accomplished by incubating SurA with BamA denatured in urea, then transferring it into a solution featuring liposomes, BamB and BamCDE,  then further incubating for 2 hours. As BamA was expressed with a his-tag, we performed a blot to determine the level of protein folding (Fig. 13). </p>
 
<p>
 
<p>
     Fig. 13
+
     <img src="https://static.igem.org/mediawiki/2018/7/73/T--Vilnius-Lithuania--Fig13_BAM_compl.png"/>
 +
</p>
 +
<p><strong>Fig. 13</strong> Western blot of BamA folding. 1U - sample 1 unboiled, 1B - sample 1 boiled, 2U - sample 2 unboiled, 2B - sample 2 boiled, L - ladder
 
</p>
 
</p>
<strong>Fig. 13</strong> Western blot of BamA folding. 1U - sample 1 unboiled, 1B - sample 1 boiled, 2U - sample 2 unboiled, 2B - sample 2 boiled, L - ladder
 
 
 
<p>As we can see from the results, after only 2 hours of incubation over 50% of BamA was processed and correctly folded, which is an indicator of proper functionality. At higher concentrations or within more enclosed environments, such as encapsulated within the liposome, efficiency is bound to increase.</p>
 
<p>As we can see from the results, after only 2 hours of incubation over 50% of BamA was processed and correctly folded, which is an indicator of proper functionality. At higher concentrations or within more enclosed environments, such as encapsulated within the liposome, efficiency is bound to increase.</p>
  

Revision as of 15:24, 8 November 2018

Design and Results

Results

Cell-free, synthetic biology systems open new horizons in engineering biomolecular systems which feature complex, cell-like behaviors in the absence of living entities. Having no superior genetic control, user-controllable mechanisms to regulate gene expression are necessary to successfully operate these systems. We have created a small collection of synthetic RNA thermometers that enable temperature-dependent translation of membrane proteins, work well in cells and display great potential to be transferred to any in vitro protein synthesis system.

invert