Difference between revisions of "Part:BBa K1604020"

Revision as of 16:55, 10 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 1. Biochemical pathway of βcarotene biosynthesis from pharnesyl di phospahate a colorless molecule naturally produced in E .coli.

FIGURE 2. Production of β-carotene. NEBβ 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. NEBβ 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.

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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 (..); BBaK173201, aracpbad (..) and BBa_K1604020 (aracpbad- β carotene) with 5 mM arabinose.

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 β carotene (A), BBa_K731201 aracpbad (B) and BBa_K1604020 β carotene device (C). NOTE This device was built using a part from the Cambridge 2009 team and the arabinose promoter from Trento 2012. The device produce β 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. Sequence and Features