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 β-carotene biosynthesis. [1]

FIGURE 1. Biochemical pathway of β-carotene. 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. Cells transformed with BBa_K731201(araC-pBAD) were used as negative control for β-carotene production. BBa_K731201(araC-pBAD) (A), BBa_K1604020 (β-carotene producer) with arabinose 5mM(B), and not induced (C). 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 50 °C.[2] Afterward they were centrifuged to recover the extracted pigments. On the left extraction in acetone of β-carotene. On the right TLC analysis of β-carotene extract from BBa_K1604020 (β-carotene producer) without arabinose 5mM (A), induced (B); and β-carotene reference (C).

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 Cary 8454. The spectra were acquired between 300 and 800 nm and blanked with acetone. UV-Vis spectra: reference β-carotene (green); BBa_K173201, control cells (violet): and BBa_K1604020 (araCpBAD + β-carotene) with 5 mM arabinose (red) and withouy induction(blue).

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1. Yeong-Su Kim, "Biotransformation of carotenoids to retinal by carotenoid 15,15′-oxygenase", Appl Microbiol Biotechnol (2010) 88:807–816

2. http://2009.igem.org/Team:Cambridge/Project/CA03 </li> Sequence and Features

   Assembly Compatibility:

   • 10
     COMPATIBLE WITH RFC[10]
   • 12
     COMPATIBLE WITH RFC[12]
   • 21
     INCOMPATIBLE WITH RFC[21]

     Illegal BamHI site found at 1144
     Illegal BamHI site found at 3185
     
   • 23
     COMPATIBLE WITH RFC[23]
   • 25
     INCOMPATIBLE WITH RFC[25]

     Illegal NgoMIV site found at 2721
     Illegal NgoMIV site found at 2851
     Illegal AgeI site found at 979
     Illegal AgeI site found at 1936
     
   • 1000
     INCOMPATIBLE WITH RFC[1000]

     Illegal SapI site found at 961