Difference between revisions of "Part:BBa K1604020"

Revision as of 09:13, 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.

FIGURE 4. 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 β carotene (A), BBa_K731201 aracpbad (B) and BBa_K1604020 β carotene device (C).

@@ Line 10: / Line 10: @@
 This device  contains the four genes necessary for  &beta;carotene  biosynthesis.
-<div style="text-align:center"><html><img src="https://static.igem.org/mediawiki/parts/3/35/Beta-carotene_pathway.jpg" style="width:60%;"></img></div></html></div>
+<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>
 <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>
+text-align:justify "><b>FIGURE. 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/3/3f/Unitn_pics_2015_Beta_car_wiki.jpg" style="width:40%;"></img></div></html></div>
-<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>
+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>
+<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>
-<div style="text-align:center"><img src="https://static.igem.org/mediawiki/parts/1/1d/Unitn_2015_Estratti_per_wiki.jpg" style="width:50%;"></div>
 <p style="width:600px; margin-left:150px; margin-bottom:60px;
-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>
+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">[[]]</div>
 <p style="width:600px; margin-left:150px; margin-bottom:60px;
-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>
+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>
-<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>
-NOTE
-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).