Part:BBa_K1604022
araC-pBAD + β-carotene + J23100 + blh
Device for the production of retinal
Usage and Biology
This device contains the five genes necessary for retinal biosynthesis. It contains the four genes responsible of βcarotene production (ctrEIBY) under the control of araC-pBAD. blh is under a costitutive promoter of the Anderson family (J23100) and encodes for the β-carotene 15-15'dioxygenase that catalizes the cleavege of a single molecule of β-carotene into two molecules of retinal.
FIGURE 1. Biochemical pathway of retinal biosynthesis in uncultured SAR86 bacteria. βcarotene is produced by pahrnesyl di phospahate a colorless molecule naturally produced in E .coli. Once βcarotene is synthetized it is cleaved in two molecules of retinal by blh.
FIGURE 2. Loss of β-carotene. NEBβ cells were cotransformed with BBa_K1604021 (in puC57) and BBa_ K1604020 (aracpBad-β carotene). Positive control was BBa_K1604020. The cells were grown up to an OD of 0.6 and induced with 5 mM of arabinose for 24 hours. BBa_K1604022 (β carotene) induced (A); cotrasformation of BBa_K1604021 and BBa_ K1604020 (B), with 5 mM arabinose (C), and with 5 mM arabinose, 5 uM FeSO4 and 10 mM of ascorbate (D); empty cells (E). Expression of blh causes the loss of the typical orange colored pellet of β carotene expressing cells.
FIGURE 3. Extraction of β carotene and retinal. NEBβ cells were cotransformed with BBa_K1604021 (in puC57) and BBa_ K1604020 (aracpBad-β carotene). BBa_K1604020 only was used for positive control. The cells were grown in 100 mL of LB and induced as described in figure 1. After 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 Agilent .... equipped with a deuterium and tungsten lamps. 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_K1604020 (β carotene) + BBa_K1604021 (blh) with Fe and ascorbate.
Our data show that there is a loss of β carotene when the cells express blh. The UV_Vis analysis confirmed the loss of β carotene (452 nm), but did not evidenced the presence of retinal (373 nm). For a more detailed characterization please check BBa_K1604022.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 9
Illegal NheI site found at 32 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2041
Illegal BamHI site found at 4082 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 247
Illegal NgoMIV site found at 3618
Illegal NgoMIV site found at 3748
Illegal AgeI site found at 1876
Illegal AgeI site found at 2833 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 1858
| n/a | araC-pBAD + β-carotene + J23100 + blh |