Part:BBa_K3711010
ACC
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1224
Illegal NheI site found at 1722
Illegal NheI site found at 1764 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 682
Illegal NgoMIV site found at 1442 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 344
Illegal BsaI.rc site found at 1342
Illegal BsaI.rc site found at 1646
Usage and Biology
Acetyl-CoA carboxylase (ACC) is a biotin enzyme that can catalyze the reaction of "acetyl-CoA+ATP+HCO3→malonyl-CoA+ADP+Pi". It exists widely in nature. ACC is a rate-limiting enzyme for ab initio synthesis of fatty acids, which catalyzes acetyl-CoA to malonyl-CoA, which eventually forms C16 acyl-CoA. ACC can be divided into multi-subunit ACC and multi-functional ACC. Polysubunit ACC exists in plants and bacteria and consists of four subunits, namely, biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP) and two subunits of carboxyltransferase (CT), α-CT and β-CT. Multifunctional ACC mostly exists in eukaryotes. ACC has been used in the drug design of obesity, diabetes and plant herbicides, and is also a target gene for some crops.
As a natural compound, curcumin is good at fighting against inflammatory and cancer. Derived from the rhizomes of some plants in the family Curphinae, Ceraceae, curcumin is a diketone compound existing in rhizoma curcumae longae for about 3% to 6%. Rarely, there is little botanic pigment with diketone structure like it. The outward appearance of it is an orange-yellow crystal powder, tastes slightly bitter and insoluble in water. In food production, it is mainly used for intestinal products, canned products, sauced products and others. Aside from cancer, curcumin could also decrease the blood fat, benefit the gallbladder, be against oxidation and according to some reports, contribute to the treatment of drug-resistant tuberculosis.
Molecular cloning
The bands of AOX1-α factor-CUS-AOX1 Terminator (3000bp) , AOX1-α factor-ACC-AOX1 Terminator (3000+bp), AOX1-α factor-4CL-AOX1 Terminator (3000+bp) and AOX1-α factor-LOX2-AOX1 Terminator (almost 5000bp) from colony PCR are identical to the theoretical lengths of 3046bp, 3619bp, 3523bp and 4528bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmid had successfully transformed into 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.
The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast.
SDS-PAGE
After confirmation from colony PCR and sequencing, we using the successfully integrated yeast for expression. At first, we try to detect our target protein in the supernatant since there is signal peptide.
Due to glycosylation modification of yeast expression, the molecular weight exhibited on SDS-PAGE will be larger than theoretical. Primary detection shows that we have laccase, 4CL and ACC bands of about 75kDa, LOX2 band of 100+kDa and DsbC+pepACS of about 40kDa, all of which is a bit larger(Laccase：57.01 kDa; 4CL：61.88 kDa; ACC：63.40 kDa; LOX2：102.88 kDa; DsbC+pepACS：31.72 kDa) but still within explainable and acceptable range, which could be evidence of successful expression.
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.
We have gained the best dye conditions of three kinds of hair dye(indigo, curcumin and lycopene) at a certain concentration. Under optimal conditions, we dyed 4-9 degrees of hair to get a series of dyeing discs. And we found that as for the three colors selected for the experiment, bleach the hair to 8 degrees could achieve a bright coloring effect.
Chart of the best condition of hair dye
| Dye/Condition | time | temperature | Dyeing aid ingredients | concentration（g/L） | comment |
| curcumin | 30min | 50℃ | Ethyl alcohol | 0.5 |
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.
Problem: The coloration rate of the curcumin aqueduct solution is low
Solution: We carried out the same dye addition and elution experiments as lycopene, and found that alum, potassium tartarate and citric acid cannot improve the coloring effect of curcumin. The data showed that curcumin is soluble in ethanol, and the optimal coloring temperature is 50 degree, so we adjusted the solvent to 33% ethanol solution. The process of coloring was conducted in the oven at 50 degrees, and the results showed that the coloring effect significantly improved.
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.
EDTA is added to the curcumin paste to prevent metal ions from interfering with the effect -- for example, Fe3 + producing a reddish-brown tint.
Curcumin dye cream
| Ingredient | Content |
| Cream matrix | 100g |
| Sodium sulfite | 0.2g |
| Absolute ethanol | 1ml |
| Isopropyl alcohol | 2ml |
| pH 6.8 phosphate buffer | 1ml |
| Solid paraffin | 1 drop or not |
| Essence | 1 drop |
| curcumin | 0.5g |
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.
| None |