Difference between revisions of "Part:BBa K1604020"

(Usage and Biology)
 
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<partinfo>BBa_K1604020 short</partinfo>
 
<partinfo>BBa_K1604020 short</partinfo>
  
This device produces &beta; carotene under the control of an arabinose promoter.
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This device produces &beta;-carotene under the control of an arabinose promoter.
 
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===Usage and Biology===
 
===Usage and Biology===
  
This device contains the four genes necessary for &beta;carotene biosynthesis.  
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This device contains the four genes necessary for &beta;-carotene biosynthesis. <html><a href="#fn:1" id="fnref:1" title="see footnote" class="footnote">[1]</a></html>
  
<div style="text-align:center"><html><img src="https://static.igem.org/mediawiki/parts/3/35/Beta-carotene_pathway.jpg"style="width:50%;"></img></div></html>
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<div style="text-align:center"><html><img src="https://static.igem.org/mediawiki/parts/c/c0/Unitn_pics_2015RetinalPathway.png"style="width:40%;"></img></div></html>
<p style="width:600px;margin: 20px auto 60px; text-align:justify"><b>FIGURE 1. Biochemical pathway of  &beta;carotene.</b> &beta;carotene biosynthesis from pharnesyl di phospahate a colorless molecule naturally produced in <i> E . coli</i>.</p>
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<p style="width:600px;margin: 20px auto 60px; text-align:justify"><b>FIGURE 1. Biochemical pathway of  &beta;-carotene.</b> &beta;carotene biosynthesis from pharnesyl di phospahate a colorless molecule naturally produced in <i> E. coli</i>.</p>
  
 
<div style="text-align:center"><html><img src="https://static.igem.org/mediawiki/parts/a/a7/Unitn_pics_2015_k20pellet.jpg"style="width:50%;"></img></div></html>
 
<div style="text-align:center"><html><img src="https://static.igem.org/mediawiki/parts/a/a7/Unitn_pics_2015_k20pellet.jpg"style="width:50%;"></img></div></html>
<p style="width:600px;margin: 20px auto 60px; text-align:justify"><b>FIGURE 2. Production of &beta;-carotene</b>. NEB10&beta; cells were transformed with BBa_K1604020 and grown up to an OD of 0.6 and induced with 5 mM of arabinose for 24 hours. BBa_K1604020 (&beta; carotene) induced (A) and not induced (B); Also uninduced cells produced high amounts of  &beta;carotene.</p>
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<p style="width:600px;margin: 20px auto 60px; text-align:justify"><b>FIGURE 2. Production of &beta;-carotene</b>. NEB10&beta; cells were transformed with BBa_K1604020 and grown up to an OD of 0.6 and induced with 5 mM of arabinose for 24 hours. Cells transformed with BBa_K731201 (araC-pBAD) were used as negative control for &beta;-carotene production. BBa_K731201 (araC-pBAD) (A), BBa_K1604020 (&beta;-carotene producer)  with arabinose 5mM (B), and not induced (C). Also uninduced cells produced high amounts of  &beta; carotene.</p>
  
 
<div style="text-align:center"><html><img src="https://static.igem.org/mediawiki/parts/5/5c/Unitn_pics_2015extraction.jpg"style="width:75%;"></img></div></html>
 
<div style="text-align:center"><html><img src="https://static.igem.org/mediawiki/parts/5/5c/Unitn_pics_2015extraction.jpg"style="width:75%;"></img></div></html>
  
 
<p style="width:600px; margin-left:150px; margin-bottom:60px; text-align:justify">
 
<p style="width:600px; margin-left:150px; margin-bottom:60px; text-align:justify">
<b>FIGURE 3. Extraction of &beta;-carotene.</b> NEB10&beta; cells were transformed with BBa_K1604020 and grown in 100 mL of LB. The cells were induced with 5 mM of arabinose and for 24 hours. The cells were span down and the supernatant was discarded. The pellets were incubated with 2.5 mL of acetone for 10 min at 50C. Afterward they were centrifuged to recover the extracted pigments. Panel A. extraction in acetone of &beta;carotene. Panel B. TLC analysis of &beta;carotene reference and BBa_K1604020 induced and uninduced with arabinose.</p>
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<b>FIGURE 3. Extraction of &beta;-carotene.</b> NEB10&beta; cells were transformed with BBa_K1604020 and grown in 100 mL of LB. The cells were induced with 5 mM of arabinose and for 24 hours. The cells were span down and the supernatant was discarded. The pellets were incubated with 2.5 mL of acetone for 10 min at 50 °C.<html><a href="#fn:2" id="fnref:2" title="see footnote" class="footnote">[2]</a></html> Afterward they were centrifuged to recover the extracted pigments. On the left extraction in acetone of &beta;-carotene. On the right TLC analysis of &beta;-carotene extract from BBa_K1604020 (&beta;-carotene producer)  without arabinose 5mM (A), induced (B); and &beta;-carotene reference (C).</p>
  
 
<div style="text-align:center"><html><img src="https://static.igem.org/mediawiki/parts/0/09/Unitn_pics_graficoUV_k1604020.jpg"style="width:75%;"></img></div></html>
 
<div style="text-align:center"><html><img src="https://static.igem.org/mediawiki/parts/0/09/Unitn_pics_graficoUV_k1604020.jpg"style="width:75%;"></img></div></html>
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<p style="width:600px; margin-left:150px; margin-bottom:60px; text-align:justify">
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<b>FIGURE 4. UV-Vis spectra of carotenoids. </b> The pigment were extracted as described in Figure 3. The extracted samples were diluted 1:7 in acetone and the spectra were taken with UV-VIS Agilent Cary 8454. The spectra were acquired between 300 and 800 nm and blanked with acetone. UV-Vis spectra: reference &#946;-carotene (green); BBa_K173201, control cells (violet):  and BBa_K1604020 (araCpBAD + &#946;-carotene) with 5 mM arabinose (red) and without induction (blue).</p>
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Check out our Wiki <html><a href="http://2015.igem.org/Team:UNITN-Trento">UNITN-Trento iGEM 2015</a>! </html>
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<div class="footnotes">
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<hr />
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<ol>
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<li id="fn:1">
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<p>Yeong-Su Kim, "Biotransformation of carotenoids to retinal by carotenoid 15,15′-oxygenase", Appl Microbiol Biotechnol (2010) 88:807–816</p>
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</li>
  
<p style="width:600px;margin: 20px auto 60px; text-align:justify"><b>FIGURE 4. UV VIS spectra of carotenoids. </b> The pigment were extracted as described in Figure 3. The extracted samples were diluted 1:7 in acetone and the spectra were taken with UV-VIS Agilent. The spectra were acquired between 300 and 800 nm and blanked with acetone.Panel B UV VIS spectra: reference &#946;carotene (..); BBaK173201, aracpbad (..) and  BBa_K1604020 (aracpbad- &#946; carotene) with 5 mM arabinose.</p>  
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<li id="fn:2">
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<p>http://2009.igem.org/Team:Cambridge/Project/CA03</p>
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</li>
  
  

Latest revision as of 17:10, 18 September 2015

araC-pBAD + beta-carotene

This device produces β-carotene under the control of an arabinose promoter.

Usage and Biology

This device contains the four genes necessary for β-carotene biosynthesis. [1]

FIGURE 1. Biochemical pathway of β-carotene. βcarotene biosynthesis from pharnesyl di phospahate a colorless molecule naturally produced in E. coli.

FIGURE 2. Production of β-carotene. NEB10β cells were transformed with BBa_K1604020 and grown up to an OD of 0.6 and induced with 5 mM of arabinose for 24 hours. Cells transformed with BBa_K731201 (araC-pBAD) were used as negative control for β-carotene production. BBa_K731201 (araC-pBAD) (A), BBa_K1604020 (β-carotene producer) with arabinose 5mM (B), and not induced (C). Also uninduced cells produced high amounts of β carotene.

FIGURE 3. Extraction of β-carotene. NEB10β cells were transformed with BBa_K1604020 and grown in 100 mL of LB. The cells were induced with 5 mM of arabinose and for 24 hours. The cells were span down and the supernatant was discarded. The pellets were incubated with 2.5 mL of acetone for 10 min at 50 °C.[2] Afterward they were centrifuged to recover the extracted pigments. On the left extraction in acetone of β-carotene. On the right TLC analysis of β-carotene extract from BBa_K1604020 (β-carotene producer) without arabinose 5mM (A), induced (B); and β-carotene reference (C).

FIGURE 4. UV-Vis spectra of carotenoids. The pigment were extracted as described in Figure 3. The extracted samples were diluted 1:7 in acetone and the spectra were taken with UV-VIS Agilent Cary 8454. The spectra were acquired between 300 and 800 nm and blanked with acetone. UV-Vis spectra: reference β-carotene (green); BBa_K173201, control cells (violet): and BBa_K1604020 (araCpBAD + β-carotene) with 5 mM arabinose (red) and without induction (blue).

Check out our Wiki UNITN-Trento iGEM 2015!




  1. Yeong-Su Kim, "Biotransformation of carotenoids to retinal by carotenoid 15,15′-oxygenase", Appl Microbiol Biotechnol (2010) 88:807–816

  2. http://2009.igem.org/Team:Cambridge/Project/CA03


  3. Sequence and Features


    Assembly Compatibility:
    • 10
      COMPATIBLE WITH RFC[10]
    • 12
      COMPATIBLE WITH RFC[12]
    • 21
      INCOMPATIBLE WITH RFC[21]
      Illegal BamHI site found at 1144
      Illegal BamHI site found at 3185
    • 23
      COMPATIBLE WITH RFC[23]
    • 25
      INCOMPATIBLE WITH RFC[25]
      Illegal NgoMIV site found at 2721
      Illegal NgoMIV site found at 2851
      Illegal AgeI site found at 979
      Illegal AgeI site found at 1936
    • 1000
      INCOMPATIBLE WITH RFC[1000]
      Illegal SapI site found at 961