Difference between revisions of "Part:BBa K1604020"

(→‎Usage and Biology)
(→‎Usage and Biology)
Line 22: Line 22:
 
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>
 
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>
  
−
<div style="text-align:center"><html><img src="" style="width:50%;"></img></div></html></div>
+
<div style="text-align:center"><html><img src="https://static.igem.org/mediawiki/parts/5/5c/Unitn_pics_2015extraction.jpg" style="width:70%;"></img></div></html></div>
 
<p style="width:600px; margin-left:150px; margin-bottom:60px;
 
<p style="width:600px; margin-left:150px; margin-bottom:60px;
−
text-align:justify "><b>FIGURE 4. HPLC analysis of carotenoids.</b> 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 &beta; carotene device (C).</p>
+
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>  
 +
 
  
  

Revision as of 09:27, 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.

FIGURE. Biochemical pathway of βcarotene. 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. BBa_K1604020 (β carotene) induced (A) and not induced (B); 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 50C. Afterward they were centrifuged to recover the extracted pigments. Panel A. extraction in acetone of βcarotene. Panel B. TLC analysis of βcarotene reference and BBa_K1604020 induced and uninduced with arabinose.