Part:BBa_K1604020
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. The samples were diluted 1:7 in acetone and the spectra were taken with UV-VIS Agilen. The spectra were acquired between 300 and 800 nm and blanked with acetone. Panel A extraction in acetone of carotenoids and retinoids. Panel B UV VIS spectra: BBa_K1604020 (β carotene) with arabinose, Fe and ascorbate (purple), BBa_K1604022 (blh + β carotene) with Fe and ascorbate.
FIGURE4. HPLC analysis of carotenoids and retinoids. The pigments 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 retinal and β carotene (A), BBa_K1604020 (β carotene) (B), BBa_K1604022 (blh).
Our data show that there is a loss of β carotene when the cells express blh. The UV_Vis analysis and the HPLC confirmed the loss of β carotene when blh is being expressed, but did not evidence the presence of retinal. We think that β carotene is being cleaved to form retinal (as shown by the evident loss of color, and the absence of a peak in the HPLC or UV-VIS spectrum), and is immediately taken by the cell to enter different biochemical pathways. The biosynthesis of retinal involves the formation of intermediates molecules that could also be used by E. coli in different metabolic reactions.
Sequence and Features
Illegal BamHI site found at 3185
Illegal NgoMIV site found at 2851
Illegal AgeI site found at 979
Illegal AgeI site found at 1936
| n/a | araC-pBAD + beta-carotene |