Part:BBa_K274120

CrtEBI under pBad promoter

This Biobrick is created by putting enzyme cassette CrtEBI (with individual rbs) of Part BBa_K274100 under arabinose-induced promoter I0500.

Enzyme cassette CrtEBI (with individual rbs) of Part BBa_K274100 converts colourless farnesyl pyrophosphate to red lycopene (via intermediates geranylgeranyl pyroiphosphate and phytoene).

Amount of lycopene produced can be measured by photospectrometer with absorbance at 475nm (lycopene extraction using acetone).

Reference

Hal Alper, et al. Construction of lycopene-overproducing E. coli strains by combining systematic and combinatorial gene knockout targets. Nature Biotechnology 23 (2005).

Nishizaki T, et al. Metabolic engineering of carotenoid biosynthesis in Escherichia coli by ordered gene assembly in Bacillus subtilis. Appl Environ Microbiol. 2007 Feb

Luke Z. Yuan, et al. Chromosomal promoter replacement of the isoprenoid pathway for enhancing carotenoid production in E. coli. Metabolic Engineering 8 (2006).

Luan Tao, et al. Isolation of chromosomal mutations that affect carotenoid production in Escherichia coli: mutations alter copy number of ColE1-type plasmids. FEMS Microbiology Letters 243 (2005)

von Lintig J, et al. Filling the gap in vitamin A research. Molecular identification of an enzyme cleaving beta-carotene to retinal. J Biol Chem. 2000 Apr 21;275(16).

Characterization by UiOslo_Norway 2019

This part was of special interest to our team. The part consists of homologs of the genes we used; crtE, crtB and crtI from Deinococcus radiodurans. BBa_K274120 has individual ribosomal binding sites for each gene. The part that we made had a single ribosomal binding site followed by all three genes in a single open reading frame because the first two genes lacked stop codons (see BBa_K2971004 and BBa_K2971010). Figure 1 shows the result of expressing BBa_K274120 in E. coli (BL21) for 24 hours. The red coloration in the induced pellet is caused by the presence of lycopene. The induced culture with BBa_K274120 produced moderate amount of lycopene.

Figure 1: Induction of BBa_K274120 in Escherichia coli (BL21)
Expression was induced with 2% arabinose
From left to right :Uninduced E. coli with empty expression vector, induced E.coli with empty expression vector, uninduced E. coli with BBa_K274120, induced E.coli with BBa_K274120.

Since the production of lycopene was so high in BBa_K274120 we tried using the culture in a simple solar cell (figure 2). The design of the solar cell is comparable to a regular dye sensitized solar cell.

Figure 2: Pelleted culture of E.coli expressing BBa_K274120 used in a simple solar cell
Expression was induced in 2% arabinose.
We did not observe any detectable current from this solar cell, but successive solar cells produced small voltages seemingly dependent on the presence of light.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  Illegal BamHI site found at 3192
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2728
  Illegal NgoMIV site found at 2858
  Illegal AgeI site found at 979
  Illegal AgeI site found at 1943
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961