FMO dimer

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 28
Illegal NgoMIV site found at 1402
Illegal AgeI site found at 1247
Illegal AgeI site found at 2621
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 1250
Illegal BsaI site found at 2624

Usage and Biology

FMOs exist in the cell as a complex with a reduced form of the prosthetic group and NADPH cofactor, readying them to act on substrates. The 4-hydroperoxyflavin form of the prosthetic group represents a transient intermediate of the monooxygenation process. The oxygenated and reduced forms of the prosthetic group help stabilize interactions with cofactor and substrate alternately to permit continuous enzyme turnover.
Whereas the enzyme–FAD and enzyme–FAD–NADPH complex structures have one dimer per unit cell of the P1 symmetry, the enzyme–FAD–methimazole complex has two. No conformational changes were evident when the three structures were compared in detail, permitting refinement with no crystallographic symmetry restraints.

Background related to indigo

The most ancient pigment known to humanity, indigo, now is popular in food, medic and dyeing industries. The pigment application of indigo could date back to at least 2,500 BC. and found on some blue hemp fabrics excavated from the Chinese MaWangDui and Egyptian mummies. One branch of Chinese Yao nationality is named after indigo as LanDianYao due to its unique technology of indigo dyeing. Among the food industry, indigo is used as edible pigment in the form of sodium sulfonate or aluminum, known as "bright blue" and bright blue aluminum lake in China, while being used mainly in its sodium sulfonate in the United States, called as "Indigo element" .

Molecular cloning

Fig1. Plasmid construction and colony PCR result of Pynr071c-α factor-FMO dimer-AOX1 Terminator transformed E.coli.

The bands of FMO dimer (less than 5000bp) and is identical to the theoretical lengths of 4600bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmid are successfully constructed.
After electrotransformed the FMO dimer into yeast, we still used Colony PCR to confirm the target gene is successfully transformed into the yeast cells.

Fig2 Colony PCR result of yeast after electroporation of FMO dimer.

The bands of FMO dimer (less than 5000bp) and is identical to the theoretical lengths of 4600bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that this target plasmid had successfully transformed into yeast.

SDS-PAGE

At first, we tried to detect the protein in the supernatant, but no results were obtained. Because we have already experienced similar problems. We extracted the total protein directly and go for a purification to tested whether it was in the cell. Actually we detected the protein this time but it was smaller than expected.

Fig3 SDS-PAGE result of FMO dimer after purification of yeast total protein extraction product through Nickel-affinity chromatography column.

Different from impure or permeate bands, the target protein located around 60kDa, smaller than the theoretical 107.52kDa, but similar to the theoretical molecular weight of FMO (53.96kDa).
This indicated that the FMO dimer was broken in the process of expression or extraction. In order to confirm whether the catalytic effect is better, we also added indole to its medium to observe its effect on indigo synthesis.

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.

Fig4. 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.

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.

Dye/Condition	time	temperature	Dyeing aid ingredients	concentration（g/L）	comment
indigo	2min	Room temperature	none	2	The color deepens significantly while dyeing for multiple times

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 indigo hair, 7 to 9 degree hair would become blue. As the dyeing time goes, the color would turn blue from an indigo color; 5 to 6 degree hair would be dyed to celadon, and 4 degree hair was still brown.

The dyeing results of indigo(room temperature,2g/L). From left to right: 9°(0.5,2,6min),8°(0.5,2,6min), 7°(0.5,2,6min)，6°(0.5,2,6min),,5°(0.5,2,6min),4°(0.5,2,6min)

Indigo:
Difficult: we can make indigo paste, but the hair does not dye well.
Solution: Indigo is a water-soluble component, and need to be oxidized to indigo after fixing to the hair. With water-in-oil paste as the matrix, indigo white can not fully enter the interior of the hair, and the oily substances in the matrix and excessive reductant prevent indigo white from oxidation in the hair, resulting in no effective coloring.
So we decided to design a timely manner in which indigo could be produced and used at the same time. Therefore, we consider that indigo dye can be produced and used in time -- the direct production of indigo by yeast, and the production of indigo solution as a dye in time.
For this idea, we dye indigo solution directly on the hair and find that it can be painted, but it can not color the hair evenly. So we designed a hair dye comb to make it possible to evenly smear indigo cryptosomes on the hair. The matching device is a timely fermentation tank, which can meet the needs of users with our engineering bacteria as raw materials, timely production and timely use of indigo white. For detailed information, please refer to Hardware part.

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.