AOX1-α factor-Lycopene-AOX1 Terminator
As a composite part, it consists of AOX1-α factor-crtE-AOX1 Terminator-AOX1-α factor-crtB-AOX1 Terminator-AOX1-α factor-crtI-AOX1 Terminator. AOX1, as a methanol induced strong promoter, will express crtE, crtB and crtI in the presence of methanol and secret them out of the cell through the signal peptide, α-factor. When the substrate, FPP, is added, these three enzymes react in order and catalyze FPP into lycopene.
Usage and Biology
crtE is derived from Erwinia, encodes Geranylgeranyl pyrophosphate synthase and participates 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 into octahydrolycopene and desaturation of octahydrolycopene into plant fluorene, β-carotene, protolycopene and lycopene. The crtE encodes Geranylgeranyl pyrophosphate synthase which synthesizes GGPP. Laboratory studies have shown that E.coli transformed with E.herbicola carotenoid synthesis gene could resist higher level of LTV radiation and phototoxic environment, indicating that the synthesis of carotenoid may be very important for the survival of E.herbicola in nature.
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.
crtI, which is derived from Erwinia, encodes octahydrolycopene dehydrogenase (PDS) and participates in the synthesis of carotenoids. The early steps of carotenoid biosynthesis pathway include the synthesis of Geranylgeranyl pyrophosphate (GGPP), the condensation of two GGPP molecules to octahydrolycopene, and then desaturating octahydrolycopene into plant fluorene, β-carotene, protolycopene and lycopene. crtI encodes octahydrolycopene dehydrogenase, which is responsible for the desaturating reaction from trans boyanic alkene to trans lycopene. The most important function of carotenoid pigments, especially carotene in advanced plants, is the protection from photooxidation. In the pathway of carotenoid synthesis, only crtI can convert octahydrolycopene to lycopene, and catalyze the desaturation from trans botanic alkene to trans lycopene. In this transformation process, lycopene was synthesized through four consecutive desaturations of intermediates of fluorene phytic acid, β-carotene and prolycopene. However, limited information can be obtained on the enzymes and genes of carotenoid biosynthesis, because the enzyme produced by crtI is immediately inactivated by separation from the membrane environment, thus preventing its purification and the subsequent cloning of the gene that encodes it.
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.
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
|Dye/Condition||time||temperature||Dyeing aid ingredients||concentration（g/L）||comment|
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.
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.
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.
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 dye cream
|pH 6.8 phosphate buffer||1ml|
|Solid paraffin||1 drop or not|
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.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 6088
Illegal NheI site found at 6517
- 21Illegal BglII site found at 6892
Illegal XhoI site found at 1187
Illegal XhoI site found at 3593
Illegal XhoI site found at 6020
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 4325
Illegal AgeI site found at 1863
- 1000Illegal BsaI site found at 4319
Illegal BsaI site found at 7278
Illegal BsaI.rc site found at 7230