Difference between revisions of "Part:BBa K1934030"
 
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<partinfo>BBa_K1934030 short</partinfo>
 
<partinfo>BBa_K1934030 short</partinfo>
  
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This part contains the sequence coding for the streptavidin protein linked to a specific cellulose binding domain, CipA protein (Cellulosomal scaffolding protein A), located in N-terminal.</p>
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<p>This part contains the sequence coding for the streptavidin protein linked to a specific cellulose binding domain, [[Part:BBa_K1615111|CipA protein - BBa_K1615111]] (Cellulosomal scaffolding protein A), located at the C-terminus.</p>
  
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Streptavidin is a 52,8 kDa protein which has the ability to bind to biotin with high affinity. Indeed, this can be explained by its structure and the formation of an extensive network of intramolecular interactions when biotin is in the binding site. This strong noncovalent link is used in many biotechnologies especially in purification and detection assays. The combination of streptavidin with other proteins thus enables to confer new properties such as a cellulose-binding activity with the integration of CBDCipA[1]. The part BBa_K1934030 was designed as a streptavidin-CipA generator to compare its cellulose binding ability with streptavidin-CBDs <a><href="https://parts.igem.org/Part:BBa_K1934020">(part BBa_K1934020)</a>.
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<p>Streptavidin is a 52.8 kDa protein which has the ability to bind to biotin with high affinity. Indeed, this can be explained by its structure and the formation of an extensive network of intramolecular interactions when biotin is in the binding site. This strong noncovalent link is used in many biotechnologies especially in purification and detection assays. The combination of streptavidin with other proteins thus enables to confer new properties such as a cellulose-binding activity with the integration of CBD-CipA <sup>[1]</sup>. The part BBa_K1934030 was designed as a streptavidin-CipA generator to compare its cellulose-binding ability with streptavidin-CBDs [[Part:BBa_K1934020|part BBa_K1934020]]. The idea was to retain the best performer in cellulose binding.</p>
  
 
[1] Bayer, E. A., Chanzy, H., Lamed, R., & Shoham, Y. (1998). Cellulose, cellulases and cellulosomes. Current opinion in structural biology, 8(5), 548-557.
 
[1] Bayer, E. A., Chanzy, H., Lamed, R., & Shoham, Y. (1998). Cellulose, cellulases and cellulosomes. Current opinion in structural biology, 8(5), 548-557.
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<html>
 
<html>
 
<h3 id="CBD">Purification Using Cellulose Affinity</h3>
 
<h3 id="CBD">Purification Using Cellulose Affinity</h3>
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<p>The BBa_K1934030 part conceived by the 2016 INSA-Lyon team and synthesized by IDT was cloned into pSB1C3 and transformed into the E. <i>coli</i> NM522 strain. One recombinant clone was grown overnight in LB at 24°C, with IPTG 1 mM and glucose 5 mM. Cells were harvested and resuspended in 1 mL lysis buffer (50 mM Tris, 300 mM NaCl, 10% glycerol). Then the mix was sonicated 5 times 30 seconds on ice at moderate power. The lysate was centrifuged at 14.000 g for 10 min. The supernatant was treated as follow:   
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<p>The BBa_K1934030 part, conceived by the 2016 INSA-Lyon team and synthesized by IDT, was cloned into pSB1C3 and transformed into the <i>E. coli</i> NM522 strain. One recombinant clone was grown overnight in LB at 24°C, with IPTG 1 mmol.L<sup>-1</sup> and glucose 5 mmol.L<sup>-1</sup>. Cells were harvested and resuspended in 1 mL lysis buffer (50 mmol.L<sup>-1</sup> Tris, 300 mmol.L<sup>-1</sup> NaCl, 10% glycerol). Then the mix was sonicated 5 times 30 seconds on ice at moderate power. The lysate was centrifuged at 14,000 g for 10 min. The supernatant was treated as follow:   
 
<ul style="list-style-type:circle">
 
<ul style="list-style-type:circle">
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   <li>Wash microcrystaline Cellulose five time in water. Then equilibrate in wash buffer (Amonium Sulfate 1M). Pack the cellulose (10x10 mm) in small chromatography columns (we used syringes barrels). </li>
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   <li>Wash microcrystalline cellulose five times in water. Then equilibrate in washing buffer (ammonium sulfate 1 mol.L<sup>-1</sup>). Pack the cellulose (10x10mm) in small chromatography columns (we used syringes barrels).</li>
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   <li> Gently pour the lysate supernatant on the column. Once the liquid starts to flow out regularly measure the OD280 of the different fractions. Continue pouring wash buffer until the OD280 stabilizes around zero. </li>
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   <li>Gently pour the lysate supernatant on the column. Once the liquid starts flowing through evenly, measure the OD<sub>280</sub> of the different fractions. Continue pouring washing buffer until the OD<sub>280</sub> stabilizes around zero.</li>
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   <li> Change the washing buffer to water. OD280 shortly rises. Keep the fractions with highest OD280. They should contain the protein. </li>
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   <li>Change the washing buffer to water. OD<sub>280</sub> shortly rises. Keep the fractions with the highest OD<sub>280</sub>. They should contain the protein.</li>
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<li> Analyse collected fractions on an SDS PAGE.  
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  <li>Analyze collected fractions on a SDS-PAGE.</li></ul>
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Optionally, proteins may be concentrated using ultrafiltration.</li>
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Optionally, proteins may be concentrated using ultrafiltration.
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</ul></p>  
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</p>  
  
  
  
  
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<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/2016/0/00/INSA-Lyon_SCipA_elution.jpeg" width = "800"/><figcaption><b>Figure 1. Purification of the chimeric Streptavidin-CipA protein on a cellulose column</b> This elution graph shows a first peak, present for both the control and our expression culture.This first peak corresponds to unbound proteins. In presence of water, only one peak was observed: it’s the elution peak of our protein.  </figcaption></figure>
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<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/2016/0/00/INSA-Lyon_SCipA_elution.jpeg" width = "800"/><figcaption><b>Figure 1. Purification of the chimeric streptavidin-CipA protein on a cellulose column.</b> This elution graph shows a first peak, present for both the control and our expression culture.This first peak corresponds to unbound proteins. In presence of water, only one peak was observed: it’s the elution peak of our protein.  </figcaption></figure>
  
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<h3 id="RT">2. BBa_K1934030 encodes a protein able to bind both biotin and cellulose</h3>
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<h3 id="RT">BBa_K1934030 encodes a protein able to bind both biotin and cellulose</h3>
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<figure><p style="text-align:center;"><img src= "https://static.igem.org/mediawiki/2016/f/f2/INSA-Lyon_principle_SCBD.jpeg" width = "400"/><figcaption>
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<figure><p style="text-align:center;"><img src= "https://static.igem.org/mediawiki/2016/f/f2/INSA-Lyon_principle_SCBD.jpeg" width = "400"/></figure>
  
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The  affinity to cellulose of the CipA-streptavidin encoded by BBa_K1934030 was compared to the one of commercial streptavidin. A molecule of Fluorescein was grafted at the 5’ end of a DNA oligo carrying a molecule of biotin at its 3’ end. This DNA oligo constitutes the reporter system. Such modified oligo was mixed either with the engineered streptavidin-CBD or with commercial Streptavidin. The resulting mix was incubated with microcrystalline cellulose in presence of PBS for 1 hour.The cellulose was then washed twice with fresh PBS and the fluorescence was measured. Every experiment was done in triplicate.  
+
The  affinity to cellulose of the streptavidin-CipA encoded by BBa_K1934030 was compared to the one of commercial streptavidin. A molecule of fluorescein was grafted at the 5’ end of a DNA oligo carrying a molecule of biotin at its 3’ end. This DNA oligo constitutes the reporter system. Such modified oligo was mixed either with the engineered streptavidin-CipA or with commercial Streptavidin. The resulting mix was incubated with microcrystalline cellulose in presence of PBS for 1 hour. The cellulose was then washed twice with fresh PBS and the fluorescence was measured. Every experiment was done in triplicate.  
  
  
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<figure><p style="text-align:center;"><img src= "https://static.igem.org/mediawiki/2016/6/64/INSA-Lyon_SCipA_linking.jpeg" width = "400"/><figcaption><b>Figure 2. The Streptavidin-CipA is able to bind biotin and cellulose.</b> The raw cellulose mixed with our report system shows no fluorescence (first bar). The measured fluorescence indicates that commercial streptavidin was able to bind our reporter system and sticks at a low extent to cellulose. We conclude that this results from none-specific adsorption. For the CBD-Streptavidin part (BBa_K1934020), a high fluorescent signal was recorded. </figcaption></figure>
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<figure><p style="text-align:center;"><img src= "https://static.igem.org/mediawiki/2016/6/64/INSA-Lyon_SCipA_linking.jpeg" width = "400"/><figcaption><b>Figure 2. Streptavidin-CipA is able to bind biotin and cellulose.</b> Raw cellulose mixed with our report system shows no fluorescence (first bar). The measured fluorescence indicates that commercial streptavidin was able to bind our reporter system and sticks at a low extent to cellulose. We concluded that it resulted from non-specific adsorption. For the streptavidin-CipA part (BBa_K1934030), a high fluorescent signal was recorded. </figcaption></figure>
 
<strong>This experiment shows that this streptavidin-CipA protein is able to bind efficiently biotin and cellulose at the same time. </strong>
 
<strong>This experiment shows that this streptavidin-CipA protein is able to bind efficiently biotin and cellulose at the same time. </strong>
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The same experiment was done for the <a><href="https://parts.igem.org/Part:BBa_K1934020">BBa_K1934020</a>: part (using two different cellulose binding domains). The binding efficiency of streptavidin-CBDs tend to be slightly lower compared to streptavidin-CipA (x1,1) but was not statistically demonstrated.  
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The same experiment was done for the <a href="https://parts.igem.org/Part:BBa_K1934020">BBa_K1934020</a> part (using two different cellulose binding domains). The binding efficiency of streptavidin-CBDs tend to be slightly lower compared to streptavidin-CipA (x1.1) but was not statistically demonstrated.  
  
 
</html>
 
</html>

Latest revision as of 18:56, 25 October 2016

Streptavidin with CBD_CipA (cellulosomal scaffolding protein A)

This part contains the sequence coding for the streptavidin protein linked to a specific cellulose binding domain, CipA protein - BBa_K1615111 (Cellulosomal scaffolding protein A), located at the C-terminus.

Streptavidin is a 52.8 kDa protein which has the ability to bind to biotin with high affinity. Indeed, this can be explained by its structure and the formation of an extensive network of intramolecular interactions when biotin is in the binding site. This strong noncovalent link is used in many biotechnologies especially in purification and detection assays. The combination of streptavidin with other proteins thus enables to confer new properties such as a cellulose-binding activity with the integration of CBD-CipA [1]. The part BBa_K1934030 was designed as a streptavidin-CipA generator to compare its cellulose-binding ability with streptavidin-CBDs part BBa_K1934020. The idea was to retain the best performer in cellulose binding.

[1] Bayer, E. A., Chanzy, H., Lamed, R., & Shoham, Y. (1998). Cellulose, cellulases and cellulosomes. Current opinion in structural biology, 8(5), 548-557.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 461
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 409

Characterization

Purification Using Cellulose Affinity

The BBa_K1934030 part, conceived by the 2016 INSA-Lyon team and synthesized by IDT, was cloned into pSB1C3 and transformed into the E. coli NM522 strain. One recombinant clone was grown overnight in LB at 24°C, with IPTG 1 mmol.L-1 and glucose 5 mmol.L-1. Cells were harvested and resuspended in 1 mL lysis buffer (50 mmol.L-1 Tris, 300 mmol.L-1 NaCl, 10% glycerol). Then the mix was sonicated 5 times 30 seconds on ice at moderate power. The lysate was centrifuged at 14,000 g for 10 min. The supernatant was treated as follow:

  • Wash microcrystalline cellulose five times in water. Then equilibrate in washing buffer (ammonium sulfate 1 mol.L-1). Pack the cellulose (10x10mm) in small chromatography columns (we used syringes barrels).
  • Gently pour the lysate supernatant on the column. Once the liquid starts flowing through evenly, measure the OD280 of the different fractions. Continue pouring washing buffer until the OD280 stabilizes around zero.
  • Change the washing buffer to water. OD280 shortly rises. Keep the fractions with the highest OD280. They should contain the protein.
  • Analyze collected fractions on a SDS-PAGE.
Optionally, proteins may be concentrated using ultrafiltration.

Figure 1. Purification of the chimeric streptavidin-CipA protein on a cellulose column. This elution graph shows a first peak, present for both the control and our expression culture.This first peak corresponds to unbound proteins. In presence of water, only one peak was observed: it’s the elution peak of our protein.

BBa_K1934030 encodes a protein able to bind both biotin and cellulose

The affinity to cellulose of the streptavidin-CipA encoded by BBa_K1934030 was compared to the one of commercial streptavidin. A molecule of fluorescein was grafted at the 5’ end of a DNA oligo carrying a molecule of biotin at its 3’ end. This DNA oligo constitutes the reporter system. Such modified oligo was mixed either with the engineered streptavidin-CipA or with commercial Streptavidin. The resulting mix was incubated with microcrystalline cellulose in presence of PBS for 1 hour. The cellulose was then washed twice with fresh PBS and the fluorescence was measured. Every experiment was done in triplicate.

Figure 2. Streptavidin-CipA is able to bind biotin and cellulose. Raw cellulose mixed with our report system shows no fluorescence (first bar). The measured fluorescence indicates that commercial streptavidin was able to bind our reporter system and sticks at a low extent to cellulose. We concluded that it resulted from non-specific adsorption. For the streptavidin-CipA part (BBa_K1934030), a high fluorescent signal was recorded.
This experiment shows that this streptavidin-CipA protein is able to bind efficiently biotin and cellulose at the same time. The same experiment was done for the BBa_K1934020 part (using two different cellulose binding domains). The binding efficiency of streptavidin-CBDs tend to be slightly lower compared to streptavidin-CipA (x1.1) but was not statistically demonstrated.