Latest revision as of 12:19, 21 October 2021

crtB coding sequence encoding phytoene synthase

This is the coding sequence of crtB from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087). It encodes phytoene synthase, part of the carotenoid biosynthesis pathway, which converts geranylgeranyl diphosphate to phytoene (Misawa, N., Nakagawa, N., Kobayashi, K., Yamano, S., Nakamura, K., and Harashima, K. 1990. Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli. Journal of Bacteriology 172, 6704-612).

Sequence and Features

HUST-China 2021 iGEM team

Usage and Biology

crtB is derived from Erwinia and encodes octahydrolycopene synthase (PSY), which is involved in the synthesis of carotenoids. The early steps of carotenoid biosynthesis pathway include the synthesis of Geranylgeranyl pyrophosphate (GGPP), the condensation of two molecules of GGPP to octahydrolycopene, and desaturation of octahydrolycopene into plant fluorene, β-carotene, protolycopene and lycopene. crtB encodes octahydrolycopene synthase, which is responsible for the condensation of two molecules of GGPP into octahydrolycopene. Carotenoids are widely found in nature. More than 630 different natural carotenoids have been identified. They are de novo synthesized from isoprene-like precursors, only in photosynthetic organisms and some microorganisms. The synthesis of carotenoids is encoded by plasmids or chromosome genes. The genes that encode carotenoid biosynthesis are clustered in a 12.4kb fragment. Genetic studies have shown that the expression of these genes requires CAMP.

Background related to lycopene

One kind of common carotenoid which is widely found in plants, lycopene is used as the red pigment. Red as it is, it looks like acicular crystal and is soluable in chloroform, benzene and fat but not in water while unstable in presence of light or oxygen and turns brown when ferrum is on its way. Molecular formula C40H56, relative molecular weight 36.85, having 11 conjugated double bonds and 2 non-conjugated double bonds, forming as a kind of hydrocarbons of straight strands. Without the biological activity of Vit-A, lycopene is a strong antioxidant. Red, matured fruit like tomato, carrot, watermelon, papaya and guava contains huge amount of lycopene which could be used as pigment in food industry and material of anti-oxidation health products.

Molecular cloning

Fig1. Colony PCR results of AOX1-α factor-FMO-AOX1 Terminator, AOX1-α factor-crtE-AOX1 Terminator, AOX1-α factor-crtB-AOX1 Terminator and AOX1-α factor-crtI-AOX1 Terminator transformed E.coli

The bands of AOX1-α factor-FMO-AOX1 Terminator (3000+bp), AOX1-α factor-crtE-AOX1 Terminator (almost 3000bp), AOX1-α factor-crtB-AOX1 Terminator (less than 3000bp) and AOX1-α factor-crtI-AOX1 Terminator (3000+bp) from colony PCR are identical to the theoretical lengths of 3214bp, 2746bp, 2767bp and 3316 bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmid had successfully transformed into E.coli

Fig2. Plasmid construction and colony PCR results of Panb1-α factor-4CL-AOX1 Terminator, Panb1-α factor-crtI-AOX1 Terminator, Panb1-α factor-crtB-AOX1 Terminator and Panb1-α factor-crtE-AOX1 Terminator transformed E.coli

The bands of Panb1-α factor-4CL-AOX1 Terminator (3000+bp), Panb1-α factor-crtI-AOX1 Terminator (3000bp), Panb1-α factor-crtB-AOX1 Terminator (2000+bp) and Panb1-α factor-crtE-AOX1 Terminator (2000+bp) from colony PCR are identical to the theoretical lengths of 3185bp, 3046bp, 2198bp and 2177bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmid are successfully constructed.
Using E.coli for amplification, we extract and digest them with Bgl I or Sal I to get linear plasmid, which could be integrated into yeast genome to avoid getting lost while being frozen. Then, concentration of linear plasmid is also applied to achieve higher copy number and higher expression level. Several rounds of electroporation later, we successfully get all the plasmid with AOX1 as promoter into yeast.

Fig3. Colony PCR result of yeast after electroporation through electrophoresis The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast.

SDS-PAGE

Fig4. SDS-PAGE result of crtB after purification of yeast total protein extraction product through Nickel-affinity chromatography column

Different from impure or permeate bands, the target protein located around 50kDa, bigger than the theoretical 35.30kDa but still within explainable and acceptable range of glycosylation modification. crtB could be confirmed as successfully expressed.

Pigment synthesis

For some of our enzymes don’t have standard protocol to estimate their activity at present, we add substrates into culturing medium accordingly to find out whether there exists active target enzymes and do get our indigo and lycopene synthesized.

Fig5. Medium for expression with substrates

From left to right: GS115 medium with indole and FPP as control; Panb1-FMO-AOX1 Terminator medium with indole; mixture of Panb1-crtE-AOX1 Terminator、Panb1-crtB-AOX1 Terminator、Panb1-crtI-AOX1 Terminator medium with FPP.

Hair dyeing experiment

We measured the standard curves of three pigments before using them for hair dyeing experiment. We also found that the amount of melanin contained in hair can have a significant effect on hair dyeing outcomes. Therefore, we define different colors of hair based on bleaching.

Chart of the best condition of hair dye

Under the best conditions, we dyed the hair from 4 degree to 9 degree, and got a series of colors. It is found that it only needed to be bleached to 8 degree so that the hair would show a bright color for all three kinds of dye.
As to lycopene hair, 8 or 9 degree hair was red, 7 degree(or below) hair was brownish, and the longer the hair was dyed, the redder it would become.

The dyeing results of lycopene（room temperature，adding alum，2g/L）。fron left to right: 9°（5,10,30min），8°（5,10,30min）, 7°（5,10,30min），6°（5,10,30min）, 5°（10,30min），4°（5,10,30min）

Problem: No literature on coloring fabrics or hair with lycopene
Solution: We conducted a gradient experiment (0.5, 1, 2, 5 g/L) to explore the effective concentration of lycopene for hair dyeing. Finally, 2g/L of lycopene is selected, at which concentration the dye fluid will not be too viscous, and has a better dyeing effect as the picture below shows.

From left to right: 0.5(30 min), 1(5, 10, 30 min), 2(5, 10, 30min), 5(5, 10, 30min)g/L of lycopene

Problem: Lycopene dye the hair with a low efficiency, a low color fastness, and a constantly discoloring process when the hair is showered by water.
Solution: We looked up the data and selected three eco-friendly color aids (alum, potassium tartrate, citric acid). Through direct color comparison and elution experiments, we found that alum can significantly improve the coloration rate and color fastness of lycopene.

From left to right, 1st-4th groups: alum（30min）、potassium tartrate（40min）、citric acid（40 min）、no color aids（40min）; 5th-8th groups: the 1st-4th hair after washing 7 times

After finishing the solution experiment, we try to mix the natural pigment into a dye that can be applied directly to the hair. At present, lycopene dye and curcumin dye with NO.1 cream matrix as carrier are obtained, and natural essence is added to improve the odor of dye paste. Indigo is an oxidizing dye with special properties, so we designed a timely fermenter. In this way, we can use our product right now when indigo is produced and reduced to indigo white.

Difficulty: when we use lycopene paste to dye hair, it is not red but orange
Solution: by analyzing the cream formula, we think what is causing this problem is sodium sulfite. We add sodium sulfite to prevent further oxidation of the pigment, but it may also reduce and fade the pigment. The solution experiment proved our conjecture. A new lycopene dye that doesn’t contain sodium sulfite found its way to red hair.

Lycopene dying cream

Color fastness test
Color fastness is an important aspect to measure the effect of dye, so we design a set of elution scheme and test the color fastness of three kinds of natural pigment dye products and the same color traditional dye paste. The results showed that the color fastness of the natural pigment dyes was better than that of the traditional dyes.