Difference between revisions of "Part:BBa K1604020"

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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.
 
 
<div style="text-align:center">[[]]</div>
 
<p style="width:600px; margin-left:150px; margin-bottom:60px;
 
text-align:justify "><b>FIGURE 1. Biochemical pathway of  &beta;carotene biosynthesis from pharnesyl di phospahate a colorless molecule naturally produced in <i> E .coli</i>. </b> </p>
 
 
 
<div style="text-align:center">[[]]</div>
 
<p style="width:600px; margin-left:150px; margin-bottom:60px;
 
text-align:justify "><b>FIGURE 2. Production of &#946;-carotene. NEB&#946; 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 (&#946; carotene) induced (A) and not induced (B); Also uninduced cells produced high amounts of  &beta;carotene. </b> </p>
 
  
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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">[[]]</div>
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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-left:150px; margin-bottom:60px;
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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>
text-align:justify "><b>FIGURE 3. Extraction of &#946; carotene. NEB&#946; 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 &#946;carotene. Panel B TLC analysis of &#946;carotene reference and BBa_K1604020 induced and uninduced with arabinose. </b> </p>
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<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: 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>
  
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<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">[[]]</div>
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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;
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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 producerwithout arabinose 5mM (A), induced (B); and &beta;-carotene reference (C).</p>
text-align:justify "><b>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 &#946;carotene (..); BBaK173201, aracpbad (..) and BBa_K1604020 (aracpbad- &#946; carotene) with 5 mM arabinose. </b> </p>  
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<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>
  
  
  
<div style="text-align:center">[[]]</div>
 
<p style="width:600px; margin-left:150px; margin-bottom:60px;
 
text-align:justify "><b>FIGURE4. HPLC analysis of carotenoids. The pigment were extracted as described before in figure 3. The samples were concentrated with N2  and methanol was added to reach a final volume of 500 uL. The sample were run on the HPLC Agilent 1100 on a Agilent Eclips XDB C8 3.5 uM (4.6  mmx150 mm) reverse phase column. Eluent used were, buffer A MeOH/H2O 7/3 + 12 mmM acetate, buffer B MeOH + 12 mmM acetate at 08 mL/min. The gradient used was 35% of buffer B up to 100% in 40 minutes. Reference with &#946; carotene (A), BBa_K731201 aracpbad (B) and BBa_K1604020 &#946; carotene device (C). </b> </p>
 
  
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<div class="footnotes">
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<hr />
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<ol>
  
  
NOTE
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<li id="fn:1">
This device was built using a part from the Cambridge 2009 team and the arabinose promoter from Trento 2012. The device produce &#946; carotene also before induction. The aracpbad promoter is demonstrated by us and others to be strictly inducible and have very low basal expression. The sequence analysis of the ctrEBIY operon shows the presence of a possible internal promoter in the sequence of the first enzyme of the pathway (ctrE). Be careful when operating this part, because the SpEI  site in the suffix is missing. The part from Cambridge 2009 had this mistake and so the error was introduced in our part. We realized that after sequencing. Other than that the device work well.
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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</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