DATA SHEET FOR BIOBRICKS Key words

Part BBa_K274100 lycopene, red, coloured pigment, carotenoid, colour output, E. coil MG1655, CrtE (geranylgeranyl Part BBa_K274110 pyrophosphate synthase), CrtB (Test in E.coli strain MG1655) (phytoene synthase), CrtI (phytoene dehydrogenase).

Basic Information Registry Entry Sequence information Length Remarks BBa_K274100 3385 bp Convert FPP to lycopene.

BBa_K274110 3448 bp Put BBa_K274100 under constitutive promoter.

Pigment Biochemistry Synthesis in E. coli Lycopene E. coli naturally synthesises Molecular formula: C40H56 IPP and DMAPP via Non- Chemical structure: mevalonate Pathway, which are readily converted to FPP (colourless). FPP is sequentially converted by Main absorbance wavelength: ~470 nm enzymes CrtE, CrtB and CrtI Natural sources: tomatoes, red carrots, to lycopene. watermelons, papayas, etc. *IPP: Isopentyl pyrophosphate. DMAPP: dimethylallyl Related compounds: carotenoid, e.g. β- pyrophosphate. carotene (see Part Bba_K274200). FPP: farnesyl pyrophosphate.

Expression in E.coli strain MG1655

Top: Cell pellet of E. coli strain MG1655 transformed with BBa_K274110 (from 200mL LB culture at 37°C for 24 hours).

Right: Growth of E. coli strain MG1655 transformed with BBa_K274110 on agar plate. Results after overnight incubation at 37°C. Small insert shows single colonies (red, indicate production of lycopene).

Photospectrometry measurement Absorbance at 474nm (lycopene) 0.50 K274110 in E.coli MG1655 0.39 0.40 E.coli MG1655 alone

0.30

0.20 Absorbance (unit) Absorbance 0.10 0.03

0.00

Absorbance at 456nm (β-carotene) and 474nm (lycopene) Lycopene in cells (pellet from 20mL LB culture at 37°C 0,50 for 24h) was extracted with 300μL acetone. For each reading, 100μL acetone exextract was diluted with 0,39 100μL water and loaded on to MicroPlate Reader for a 0,40 full specctrum scan (300nm to 800nm). Each graph 0,33 shows the average of two readings. Value normalised 0,30 with blank (50% acetone) and OD600.

0,20 Reference

Hal Alper, et al. Construction of lycopene-overproducing Absorbance (unit) Absorbance E. coli strains by combining systematic and 0,10 combinatorial gene knockout targets. Nature Biotechnology 23 (2005). 0,00 456 474 Wavelength (nm)