In our project, we developed a capsule containing two catalytic enzymes dextransucrase and inulosucrase that are coded by genes EC.2.4.1.5 and EC.2.4.1.9 to catalyse the conversion from sucrose to two types of soluble dietary fibres (SDFs) dextran and inulin respectively as shown in Figure 1. Therefore, individuals may enjoy their diets without change in flavor while reducing the human body's effective absorption of sugars in food and increasing fiber intake. With no change to the sugar content of the food, this initiative intends to promote bowel movement, regulate gut bacteria, and lower the quantity of sugar absorbed by the human body into the bloodstream.

In this part, we will demonstrate the design of our plasmid, how it was built, how to test its function and what we learned through this construction.

Design 1:

EC.2.4.1.5 belongs to the Glucansucrase (GS) family(Fig 1). It breaks sucrose molecules, polymerizes the D-glucose fraction, synthesizes α-glucan with different bond compositions, branches, and chain lengths, and releases fructose molecules. With the particular catalytic property, recombinant proteins are obtained by heterologous recombination to explore the catalytic properties of recombinant proteins for converting sucrose to produce Dextran, a dietary fiber.

Fig.1 Target gene map of EC.2.4.1.5

 

The pET-28a plasmid is a prokaryotic high-efficiency expression vector containing the anti-kanamycin gene, commonly used as a fusion protein in bacteria. The host cell induces the expression of this gene by providing T7 RNA polymerase. The pET-28a composes a new vector with EC.2.4.1.5 in the construction, and it can be extracted from Escherichia coli (E. coli).

Fig.2 pET28a plasmid

 

The template plasmid was used to obtain the EC.2.4.1.5 gene through polymerase chain reaction (PCR), and the pET28a plasmid was extracted from E. Coli. The target gene and pET28a plasmid were double-digested, ligated, and transformed into E. coli DH5α. PCR and sequenced then identified the bacterial liquid and confirmed the construction of a recombinant plasmid. The final plasmid is the aim of our construction. We used Snapgene to create the profile of the plasmid.

 

Fig.3 pET28a-EC.2.4.1.5 plasmid

 

Build 1:

The biotechnology company GeneScript synthesizes the DNA molecules containing our desired gene. Then, they are cut with restriction endonucleases Nde1 and Xho1 and amplified using polymerase chain reaction (PCR). The gene's length is 4400 bp. The bands representing EC.2.4.1.5 successfully appear at their corresponding positions in the gel, as shown in Fig 10, indicating that the cutting and amplifying are successful.

Fig.4 Double digestion of EC.2.4.1.5

 

A double digestion experiment can change the vector from a ring to a line, using Nde1 and Xho1 to cut and link the sites at the gaps at both ends of the vector.

 

               

Fig.5 Double digestion of pET28a

 

Nde1 and Xho1 also cut pET28a to make complementary sticky ends. EC.2.4.1.5 is connected to the linear plasmids with T4 ligase. Then, the heat shock conversion of the recombinant plasmids is applied to competent E. coli DH5α. We then incubated them overnight at 37°C after streak inoculating them on LB solid medium plates that included appropriate antibiotics (LB-kana), as shown in Fig 11B. Afterward, we picked three colonies from each petri dish and extracted their plasmids. PCR and gel electrophoresis (Fig 6A) were run to confirm the extracted plasmids were the ones we required. EC.2.4.1.5 (4440 bp long) appears at its corresponding position. Then, the pET28a-EC.2.4.1.5 plasmids are sent to Azenta Life Sciences for sequencing, whose results further proved our success in constructing the plasmid (Fig 7).

 

Fig.6 A: PCR identification; B: colony identification

 

After obtaining the PCR results, it was confirmed that the bands after its PCR was completed were consistent with the expected target bands, so we concluded that the recombinant plasmid was successfully constructed.

The recombinant plasmid was sent to Azenta Life Sciences for sequencing, and the sequencing results accurately proved that the pET28a-EC2.4.1.5 plasmid was constructed successfully, as seen in the DNA sequencing graph.

Fig.7 Sequencing diagram

 

The verified plasmids were transformed into E.coli BL21(DE3), incubated on LB solid medium plates (Kana+), and cultured at 37°C overnight. Four colonies were selected for PCR amplification to confirm success in the second transformation (Fig 8) and then transferred into 1L fresh LB (Kana+) culture medium for the scale-up cultivation. IPTG (0.2 mM) was used to induce the expression of genes EC.2.4.1.5 with OD600 around 0.6-0.8 and cultured at 16℃ for 20h. The proteins were then extracted from the supernatant of the E.coli BL21(DE3) after ultrasonic cell disruption and centrifugation.

Fig. 8 DNA gel electrophoresis of EC.2.4.1.5 (A) and clones of E.coli BL21(DE3) containing the gene (B)

 

Test 1:

After transforming the recombinant plasmid into BL21(DE3), we allowed it to express the protein in significant quantities by expanding the culture and IPTG induction. The protein size on the protein strip after sonication and fragmentation is 167kDa, which is consistent with the size of our target gene EC.2.4.1.5. This indicates that our target protein was correctly expressed in large quantities.

 

Fig.9 Results of SDS-PAGE of supernatant, flowthrough, wash, and elution parts for the protein purification of EC.2.4.1.5

 

Learn 1:

Problem	Solution
How to find a proper method to identify the function of the obtained enzyme	We evaluated the available equipment and materials, read relevant essays, and researched information. Finally, according to the teacher’s suggestion, we used thin-layer chromatography.
Why the results of SDS-PAGE were not clear	We may need to purify the protein by reconcentrating it. If the solution is too dilute, the results might not be precise.

Design 2:

 EC.2.4.1.9 codes for the enzyme inulosucrase, which transfers a fructose group from the disaccharide sucrose to a growing inulin chain, a fiber, to produce glucose and inulin. The pET28a plasmid has other desirable features that make it an ideal vector besides the T7 promoter and terminator it possesses. It has multiple restriction sites for the insertion of a new gene. Meanwhile, it has a sequence coding for a 6×His tag between the T7 promoter and terminator, so the protein produced can be purified via nickel column affinity chromatography.

Fig.10 The gene map of EC.2.4.1.9

 

In our project, this enzyme reduces glucose absorption by catalyzing this reaction using sucrose as its substrate, lowering the chance of getting obese or contracting non-communicable diseases. The template plasmid was used to obtain the EC.2.4.1.9 gene through polymerase chain reaction (PCR), and the pET28a plasmid was extracted from E. Coli. The target gene and pET28a plasmid were double-digested, ligated, and transformed into E. coli DH5α. PCR and sequenced then identified the bacterial liquid and confirmed the construction of a recombinant plasmid. The final plasmid is the aim of our construction. We used Snapgene to create the profile of the plasmid.

Fig. 11 Plasmid map of pET28a-EC.2.4.1.9

 

Build 2:

The biotechnology company GeneScript synthesizes the DNA molecules containing our desired gene. Then, they are cut with restriction endonucleases Nde1 and Xho1 and amplified using polymerase chain reaction (PCR). The gene's length is 2400 bp. The bands representing EC.2.4.1.9 successfully appear at their corresponding positions in the gel, as shown in Fig 19, indicating that the cutting and amplifying are successful.

 

Fig.12 The results of gel electrophoresis of EC.2.4.1.9

 

Nde1 and Xho1 are also used to cut pET28a to make complementary sticky ends. EC.2.4.1.9 is connected to the linear plasmids with T4 ligase. Then, the heat shock conversion of the recombinant plasmids is applied to competent E. coli DH5α. We then incubated them overnight at 37°C after streak inoculating them on LB solid medium plates that included appropriate antibiotics (LB-kana), as shown in Fig 20B. Afterward, we picked three colonies from each petri dish and extracted their plasmids. PCR and gel electrophoresis (shown in Fig 13A) were run to confirm the extracted plasmids were the ones we required. EC.2.4.1.9 (2400 bp long) appears at its corresponding position.

 

Fig.13 A: Gel electrophoresis to confirm pET28a-EC.2.4.1.9 plasmids. B: pET28a-EC.2.4.1.9 containing strain clones.

 

Then, the pET28a-EC.2.4.1.9 plasmids are sent to Azenta Life Sciences for sequencing, whose results further proved our success in constructing the plasmid (Fig 14).

Fig.14 The DNA sequencing diagram for pET28a-EC.2.4.1.9

 

The verified plasmids were transformed into E.coli BL21(DE3), incubated on LB solid medium plates (Kana+), and cultured at 37°C overnight. Four colonies were selected for PCR amplification to confirm success in the second transformation (Fig 15) and then transferred into 1L fresh LB (Kana+) culture medium for the scale-up cultivation. IPTG (0.2 mM) was used to induce the expression of genes EC.2.4.1.9 with OD600 around 0.6-0.8 and cultured at 16℃ for 20h. The proteins were then extracted from the supernatant of the E.coli BL21(DE3) after ultrasonic cell disruption and centrifugation.

Fig. 15 DNA gel electrophoresis of EC.2.4.1.9 (A) and clones of E.coli BL21(DE3) containing the gene (B).

 

Test 2:

Nickel affinity chromatography effectively purifies inulosucrase because the protein contains a 6×His tag. We got a more precise result with little interference by non-specifically bound proteins. Fig 8 shows only one band with a molecular weight of 88 kDa. This demonstrates that inulosucrase is successfully expressed and purified.

Fig.16 Results of SDS-PAGE of supernatant, flowthrough, wash, and elution parts for the protein purification of EC.2.4.1.9

 

Learn 2:

Problem	Solution
How to determine the ratio of two enzymes	Further validation will be conducted via HPLC.
Why the results of SDS-PAGE were not clear	We may need to purify the protein by reconcentrating it. If the solution is too dilute, the results might not be precise.