Part:BBa_K3998000

flr

flr

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.

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

Construct design

The target protein expression fragment（BBa_K3998000） is constructed into a vector of pET28a. According to Yang, G., et al. (2021), flavone reductase (FLR) discovered from Flavonifractor plautii ATCC 49531 (originally assigned as Clostridium orbiscindens DSM 6740) plays a key step in catalyzing flavonoid. Thus, we plan to over-express FLR in Eco. li (BL21) and test its function in degrading flavonoid. 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 protein expression box..

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

Experimental approach

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

Figure4 Flow chart of the engineered strain design..

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.

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