Difference between revisions of "Part:BBa K3998000"

Revision as of 04:30, 19 October 2021

flr

flr

Profile

Name: flr

Base Pairs: 6275bp

Origin: Synthesis from a genetic company

Properties: A protein used to improve the degradation of flavonoids

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.

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.

Improvement of an existing part

1.Both of the research focus on promoting the degradation of metabolites in the body by building engineering bacteria. Compared to the old part BBa_K3584002, they aimed to engineer probiotics that can be consumed by the patient, express the target gene into enzymes to degrade p-cresol, the precursor of pCS. In our project, we put FLR gene into E. coli to produce a strain secreting FLR enzyme. The strain degrades flavonoids to produce DAT and stimulate the immune system to achieve the purposes of anti-inflammatory, antibacterial and anti-cancer. Both of our research objectives are to promote the degradation of metabolites in the body by building engineering bacteria.

2. About plasmid construction, our improvement is reflected in the construction of recombinant plasmids that express different functions using the same promoter-terminator model, composite part T7 pro-tag-Lac ope-XX-T7 ter.

Figure7 Map of composite part T7 pro-tag-Lac ope-Flr-T7 ter..

Compared to the old part BBa_K3584002, composite part T7 pro-tag-Lac ope-PPO-T7 ter., we design a new part BBa_K3998000 which replaced the PPO fragment with the Flr gene fragment. Flr enzyme was successfully produced by transformed Escherichia coli. On the basis of purified recombinant Flr, an excellent Flr activator was obtained. After we obtained the purified protein Flr, namely the FLR enzyme, we conducted enzyme activity tests by using 4 kinds of flavonoids 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 gain more data to ensure the credibility of the result of our experiment. All these results indicate that our enzyme has high activity and is capable of degrading multiple flavonoids with a certain universality.

3.Future Plan

Since the FLR enzyme showed a great ability to decompose flavonoids, we are looking forward to testing how the human body would consume with and without the FLR enzyme. What’s more, if the further experiment succeeds, we will probably try to add the FLR engineered bacteria into yogurt. Yogurt contains a large number of lactic acid bacteria, which themselves belong to acidic substances. After use, they can promote gastrointestinal peristalsis and food digestion. Yogurt helps in digesting food and FLR helps in digesting flavonoids. By adding it, we could make a health-care yogurt which is great for human health improvement.

References

1. Gaohua Yang, Sen Hong, Pengjie Yang, et al. Discovery of an ene-reductase for initiating flavone and flavonol catabolism in gut bacteria, Nature communcations (2021).

2. 黄瑶. 黄酮类物质改善认知功能障碍作用机制的研究进展[J]. 养生大世界 2021年4期, 241-242页, 2021.

3. Thilakarathna S H, Rupasinghe HP. Flavonoids Bioavailability and attempts for bioavailability enhancement[J].Nutrients, 2013, 5(9):3367-3387

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