Part:BBa_K3998004

flr-chi-enoR-phy

Usage and Biology

In this project, we constructed a composite part BBa_K3998004, which is the coding sequence of protein flr-chi-enoR-phy. This fusion protein can improve the degradation of flavonoids, further produce DAT and stimulate the immune system of the human body, so as to achieve anti-inflammatory, antibacterial, anti-cancer and other purposes, at the same time to help reducing clinical treatment costs.

Figure1 Principles of the degradation of intestinal bacteria flavonoids: Flavonoids - > dihydroflavones - > charone - > dihydrocarboncarbonone.

Construct design

The target protein expression fragment（BBa_K3998004） is constructed into a vector of pET28a. The composite part is transcripted by the T7 promoter and stopped by the T7 terminator. Meanwhile, a His protein tag is inserted for future protein purification. We carried out molecular biology experiments and successfully constructed the composite part above in the vector of Eco. Li (BL21).

Figure2 Map of peT-28a-flr-chi-enoR-phy..

BBa_K3998000

Usage and Biology

In this project, we put the FLR gene into E. coli to produce a strain secreting FLR enzyme efficiently. This strain can better express the FLR gene, improve the degradation of flavonoids, further produce DAT and stimulate the immune system of the human body, so as to achieve anti-inflammatory, antibacterial, anti-cancer and other purposes, at the same time to help reducing clinical treatment costs.

Figure3 Schematic map of pET 28a and Flr expression plasmids..

BBa_K3998001

Usage and Biology

In this project, we put the chi gene into E. coli to produce a strain secreting chi enzyme efficiently. Protein of Chi convert dihydroflavone into chalcone to improve the degradation of flavonoids, further produce DAT and stimulate the immune system of the human body, so as to achieve anti-inflammatory, antibacterial, anti-cancer and other purposes, at the same time to help reducing clinical treatment costs.

BBa_K3998002

Usage and Biology

In this project, we put the enoR gene into E. coli to produce a strain secreting enoR enzyme efficiently. Protein of enoR convert chalcone into dihydrochalcone to improve the degradation of flavonoids, further produce DAT and stimulate the immune system of the human body, so as to achieve anti-inflammatory, antibacterial, anti-cancer and other purposes, at the same time to help reducing clinical treatment costs.

BBa_K3998003

Usage and Biology

In this project, we put the phy gene into E. coli to produce a strain secreting phy enzyme efficiently. Protein of phy convert dihydrochalcone into degradation products to improve the degradation of flavonoids, further produce DAT and stimulate the immune system of the human body, so as to achieve anti-inflammatory, antibacterial, anti-cancer and other purposes, at the same time to help reducing clinical treatment costs.

Figure4 Schematic map of pET 28a and Flr/chi/enoR/phy expression plasmids..

Experimental approach

In lab, we successfully constructed the plasmid and it was proved by colony PCR and sequencing result.

Preparation of pET28a vector: The vector was obtained from our plasmid library.

Acquisition of Inserts: Introducing homologous sequences of pET28a vector into 5’-end of Forward (F) & Reverse (R) primer, respectively, aiming to make the ends of amplified inserts and vectors identical to each other.

table1..

Recombination: Calculated the amount of DNA for recombination by formula. Diluted pET28a vector and inserts before recombination to make sure the loading accuracy.

table2..

Transformation

Place the competent cells on ice (i.e. DH5α competent strain). 2.Pipet 10 μl of the recombination products to 100 μl of the competent cells, flip the tube several times to mix thoroughly (DO NOT VOTEX!), and then place the tube still on ice for 30 min. The volume of transformation products should not be more than 1/6 of the volume of competent cells. 3. Heat-shock the tube at 42℃ for 45 sec and then immediately chill on ice for 2 - 3 min. 4. Add 900 μl of LB medium (without antibiotics) to the tube. Then, shake at 37℃ for 1 hour at 200 - 250 rpm. 5. Preheat the LB plate which contains appropriate selection antibiotic at 37℃ . 6. Centrifuge the culture at 5,000 rpm for 5 min, discard 900 μl of supernatant. Then, re-suspend the pellet with 100 μl of remaining medium and plate it on an agar plate which contains appropriate selection antibiotic. 7. Incubate at 37℃ for 12 -16 hours.

we successfully constructed the plasmid and it was proved by colony PCR and sequencing result.

Proof of function

Enzyme Activity Test of FLR

After obtaining the purified protein containing FLR enzyme, we tested its effectiveness in degrading flavonoid. We conducted enzyme activity tests by using 4 kinds of flavonoid samples: apigenin, chrysin, luteolin, diosmetin with the initial concentration 10mg/L and the concentration of FLR enzyme was 1mM/L. Each sample was guaranteed three replicates of the enzyme activity test in order to to ensure the credibility of the test results. The test results are listed as follows:

Figure5 Bar graph of the enzyme activity test results..

Results show that the concentration of these 4 flavonoids remarkably decreases after 2 hours which were degraded by the enzyme, even there were only 0.3-5% left after 6 hours. All these results indicate that our enzyme has high activity and is capable of degrading multiple flavonoids with a certain universality.

Bacteria Activity Test

Figure6 Bar graph of the bacteria activity test results..

All these flavonoids were almost degraded after 2 hours with only 1-7% left, which indicates the strong ability of our engineered E. coli to degrade flavonoids as we expected.

Above all, we could come to the conclusion that the FLR enzyme, as well as the engineered E. coli, is capable of degrading various flavonoids with a certain universality and practicability. In comparison, the degradability of FLR enzyme to these flavonoids could be ranked from greatest to least as apigenin > chrysin > luteolin > diosmetin.

References

4. Ravishankar D, Rajora A K, Greco F, et al. Flavonoids as prospective compounds for anti-cancer therapy[J].Int J Bio- chem Cell Biol, 2013, 45(12):2821-2831

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2742
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 469
  Illegal NgoMIV site found at 637
  Illegal NgoMIV site found at 1311
  
• 1000
  COMPATIBLE WITH RFC[1000]