Difference between revisions of "Part:BBa K274120"

Latest revision as of 01:29, 22 October 2019

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. There is some slight coloration in the pelleted culture of uninduced BBa_K274120, indicating some leaky expression in this part.

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.

We tried various methods to monitor the production of lycopene in the cells. We grew induced 200µl cultures on a microplate at 37°C, doing periodic measurements of cell count (OD600) and lycopene (OD472). Evaporation was prevented by applying a membrane to the plate that was permeable to oxygen. Unfortunately, when induced cultures were compared to empty vector controls there was not any difference in the lycopene measurement. Lysing pelleted cultures in acetone to extract lycopene proved to be inconsistent. When lycopene extracts were applied to a TLC plate (figure 3) the size of the carotenoid spot was inconsistent with the observed color of the pellet.

Figure 3: TLC on lycopene extract of induced cell cultures
The right image shows the TLC plate under UV light. The left image shows the same plate in normal light. Extracts were taken from induced BBa K274120 (furthest left on the plate), induced empty vector (middle of plate) and induced Ba K2971004 (right on the plate). There is a clear red spot at the top of the mobile phase from the sample isolated from a K2971004 confirming the presence of lycopene.

Sequence and Features