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 β-carotene production (ctrEIBY) under the control of araC-pBAD promoter. 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. [1]

FIGURE 1. Biochemical pathway of retinal biosynthesis in uncultured SAR86 bacteria. [2] 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 transformed with BBa_K1604022, and grown up to an OD of 0.6 and induced with 5 mM of arabinose for 24 hours. Cells containing BBa_K1604020 were used as potive control for β-carotene production. BBa_K1604020 (β-carotene) not induced (A); BBa_K1604022 not induced (B), with 5 mM arabinose (C), and with 5 mM arabinose, 5 uM FeSO4 and 10 mM of ascorbate (D), BBa_K731201 cells, negative control (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 transformed with BBa_K1604020 and BBa_ K1604022 were grown in 100 mL of LB and induced as described in figure 2. 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 Cary 8454 equipped with a deuterium and tungsten lamps. The spectra were acquired between 300 and 800 nm and blanked with acetone.UV-Vis spectra: β-carotene reference (violet), retinal reference (red), BBa_K1604022 (blh + β carotene) with with 5 mM arabinose 5 uM FeSO₄ and 10 mM of ascorbate in blue, BBa_K731201 control cells (green)

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 Eclipse 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 producer) (B), and BBa_K1604022 (blh expressing cells).

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.

Check out our Wiki UNITN-Trento iGEM 2015!

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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. Martinez, A.,"Proteorhodopsin Photosystem Gene Expression Enables Photophosphorylation in a Heterologous Host" Proceedings of the National Academy of Sciences 104.13 (2007): 5590-595. Web.





Sequence and Features




Assembly Compatibility:

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

  Illegal NheI site found at 9
  Illegal NheI site found at 32
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2041
  Illegal BamHI site found at 4082
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE 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
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 1858