Difference between revisions of "Part:BBa K1604020"

(Usage and Biology)
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===Usage and Biology===
 
===Usage and Biology===
  
This device contains the four genes necessary for βcarotene 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>
 
<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>
<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>
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<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 50 °C. Afterward they were centrifuged to recover the extracted pigments. Panel A. extraction in acetone of &beta;-carotene. Panel B. TLC analysis of &beta; extract from BBa_K1604020 (&beta;-carotene producer)  without arabinose 5mM (A), and induced (B) &beta;-carotene reference (C).
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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. Panel A. extraction in acetone of &beta;-carotene. Panel B. TLC analysis of &beta; extract from BBa_K1604020 (&beta;-carotene producer)  without arabinose 5mM (A), and induced (B) &beta;-carotene reference (C).
  
 
<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>
 
<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 (green); BBa_K173201, control cells (violet):  and BBa_K1604020 (aracpbad- &#946;-carotene) with 5 mM arabinose (red) and withouy induction(blue).</p>  
 
<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 (green); BBa_K173201, control cells (violet):  and BBa_K1604020 (aracpbad- &#946;-carotene) with 5 mM arabinose (red) and withouy 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>
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<li id="fn:2">
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<p>http://2009.igem.org/Team:Cambridge/Project/CA03
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</li>
  
  

Revision as of 17:53, 16 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 βcarotene biosynthesis. html><a href="#fn:1" id="fnref:1" title="see footnote" class="footnote">[1]</a></html>

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) without arabinose 5mM(B), and not induced (C). Also uninduced cells produced high amounts of βcarotene.

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. Panel A. extraction in acetone of β-carotene. Panel B. TLC analysis of β extract from BBa_K1604020 (β-carotene producer) without arabinose 5mM (A), and induced (B) 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. The spectra were acquired between 300 and 800 nm and blanked with acetone.Panel B UV-Vis spectra: reference βcarotene (green); BBa_K173201, control cells (violet): and BBa_K1604020 (aracpbad- β-carotene) with 5 mM arabinose (red) and withouy induction(blue).</p> Check out our Wiki UNITN-Trento iGEM 2015!




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