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             <caption>Figure 1: Plasmid maps of pET28a-EC.2.4.1.9</caption>
 
             <caption>Figure 1: Plasmid maps of pET28a-EC.2.4.1.9</caption>
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         <img src="https://static.igem.wiki/teams/5093/bba-k5093003/2.png" width="50%" alt="Figure 2: The gene map of EC 2.4.1.9">
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             <caption>Figure 2: The gene map of EC 2.4.1.9.</caption>
 
             <caption>Figure 2: The gene map of EC 2.4.1.9.</caption>
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             <caption>Figure 5: The results of gel electrophoresis of EC 2.4.1.9</caption>
 
             <caption>Figure 5: The results of gel electrophoresis of EC 2.4.1.9</caption>
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         <img src="https://static.igem.wiki/teams/5093/bba-k5093003/7.png" width="50%" alt="Figure 7: The DNA sequencing diagram for pET28a-EC 2.4.1.9.">
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         <img src="https://static.igem.wiki/teams/5093/bba-k5093003/7.png" width="70%" alt="Figure 7: The DNA sequencing diagram for pET28a-EC 2.4.1.9.">
 
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             <caption>Figure 7: The DNA sequencing diagram for pET28a-EC 2.4.1.9.</caption>
 
             <caption>Figure 7: The DNA sequencing diagram for pET28a-EC 2.4.1.9.</caption>
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         <img src="https://static.igem.wiki/teams/5093/bba-k5093003/9.jpg" width="50%" alt="Figure 9: SDS-PAGE of inulosucrase from EC 2.4.1.9-containing E.coli BL21(DE3).">
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         <img src="https://static.igem.wiki/teams/5093/bba-k5093003/9.jpg" width="30%" alt="Figure 9: SDS-PAGE of inulosucrase from EC 2.4.1.9-containing E.coli BL21(DE3).">
 
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             <caption>Figure 9: SDS-PAGE of inulosucrase from EC 2.4.1.9-containing E.coli BL21(DE3).</caption>
 
             <caption>Figure 9: SDS-PAGE of inulosucrase from EC 2.4.1.9-containing E.coli BL21(DE3).</caption>
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             <caption>Figure 10: Results of thin-layer chromatography. 1: The recombinant enzyme solution with a ratio of 1:1 between dextransucrase and inulosucrase; 2: The recombinant enzyme solution with a ratio of 1:2 between dextransucrase and inulosucrase; 3: The recombinant enzyme solution with a ratio of 2:1 between dextransucrase and inulosucrase.</caption>
 
             <caption>Figure 10: Results of thin-layer chromatography. 1: The recombinant enzyme solution with a ratio of 1:1 between dextransucrase and inulosucrase; 2: The recombinant enzyme solution with a ratio of 1:2 between dextransucrase and inulosucrase; 3: The recombinant enzyme solution with a ratio of 2:1 between dextransucrase and inulosucrase.</caption>
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             <caption>Figure 11: 3D mapping of glucose (right) and sucrose (left) concentrations in various conditions under 37°C.</caption>
 
             <caption>Figure 11: 3D mapping of glucose (right) and sucrose (left) concentrations in various conditions under 37°C.</caption>

Latest revision as of 05:28, 29 September 2024

pET28a-EC.2.4.1.9


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 4402
    Illegal BglII site found at 5222
    Illegal BglII site found at 5230
    Illegal XhoI site found at 6816
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2622
    Illegal NgoMIV site found at 2782
    Illegal NgoMIV site found at 4370
    Illegal NgoMIV site found at 5180
  • 1000
    COMPATIBLE WITH RFC[1000]


EC 2.4.1.9 Gene Documentation

Construction Design

This engineered plasmid comprises a pET28a backbone (BBa_K3521004) and the gene EC 2.4.1.9 (BBa_K5093001).

Figure 1: Plasmid maps of pET28a-EC.2.4.1.9
Figure 1: Plasmid maps of pET28a-EC.2.4.1.9

Essential parts in the plasmid

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 reaction is illustrated in Figure 3.

In our project, this enzyme reduces the absorption of glucose by catalyzing this reaction using sucrose as its substrate, hence lowering the chance of getting obese or contracting non-communicable diseases such as hyperglycemia, dyslipidemia, diabetes, coronary heart disease, non-alcoholic fatty liver disease, and stroke [1–15], without diminishing the sweetness of food and drink.

Its product, inulin, is a soluble dietary fiber that the gut microbiota can efficiently ferment. The high diversity of the gut microbiota has been proven to reduce the incidence of many chronic diseases, including inflammatory bowel disease, colorectal cancer, obesity, and T2DM [16–18]. Furthermore, dietary fiber could lower blood lipid concentration, reducing the risk of hyperlipidemia [18].

Figure 2: The gene map of EC 2.4.1.9
Figure 2: The gene map of EC 2.4.1.9.
Figure 3: The formation of inulin with inulosucrase from sucrose
Figure 3: The formation of inulin with inulosucrase from sucrose [18]

T7 Promoter (BBa_K3521002) and T7 Terminator (BBa_K3521002)

The T7 promoter and terminator originated in the T7 bacteriophage. The T7 promoter has a high affinity to its specific polymerase, the T7 RNA polymerase, which has a high transcription rate. When Isopropyl β-D-1-thiogalactopyranoside (IPTG), a lactose-like substance that bacteria cannot metabolize, is added, it binds to the Lac repressor on the operator to cause a conformation change, so the repressor is detached from the operator. T7 RNA polymerase can bind to the T7 promoter to initiate a fast transcription. This is very beneficial in the heterologous expression because the induction by IPTG can endure a high protein production rate.

Figure 4: An illustration of the T7 promoter and terminator
Figure 4: An illustration of the T7 promoter and terminator

Plasmid Backbone: pET28a (BBa_K3521004)

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×histidine tag between the T7 promoter and terminator, so the protein produced can be purified via nickel column affinity chromatography. Another pronounced feature is the kanamycin resistance gene (KanR). When the target protein is made, this gene on the same plasmid is also expressed so that the bacteria containing the plasmid can survive under kanamycin. This essentially facilitates the preliminary selection of successfully transformed bacteria on kanamycin-containing media because the ones that fail to take in the plasmids are killed and unable to reproduce into clones.

Experimental Approach

1. Obtaining, Amplifying, and Identifying the Gene

The biotechnology company GeneScript synthesizes the DNA molecules containing our desired gene. Then, they are cut with restriction endonucleases NdeI and XhoI 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 Figure 5, indicating that the cutting and amplifying are successful.

Figure 5: The results of gel electrophoresis of EC 2.4.1.9
Figure 5: The results of gel electrophoresis of EC 2.4.1.9

2. Plasmid Construction and Transformation

NdeI and XhoI 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 Figure 6B.

Afterwards, we picked three colonies from each petri dish and extracted their plasmids. PCR and gel electrophoresis (shown in Figure 6A) 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. 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 (Figure 7).

Figure 6: A: Gel electrophoresis to confirm pET28a-EC 2.4.1.9 plasmids. B: pET28a-EC 2.4.1.9 containing strain clones.
Figure 6: A: Gel electrophoresis to confirm pET28a-EC 2.4.1.9 plasmids. B: pET28a-EC 2.4.1.9 containing strain clones.
Figure 7: The DNA sequencing diagram for pET28a-EC 2.4.1.9.
Figure 7: The DNA sequencing diagram for pET28a-EC 2.4.1.9.

3. Protein Expression

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 (Figure 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.2.4.1.9 with OD600 around 0.6-0.8 and cultured at 16°C for 20 hours. The proteins were then extracted from the supernatant of the E.coli BL21(DE3) after ultrasonic cell disruption and centrifugation. The size of inulosucrase is 88 kDa, as confirmed in the SDS-PAGE in Figure 9.

Figure 8: DNA gel electrophoresis of EC 2.4.1.9 (A) and clones of E.coli BL21(DE3) containing the gene (B).
Figure 8: DNA gel electrophoresis of EC 2.4.1.9 (A) and clones of E.coli BL21(DE3) containing the gene (B).

4. Protein Purification

Nickel affinity chromatography effectively purifies inulosucrase because the protein contains a 6×his tag. We obtained a more precise result with little interference from non-specifically bound proteins. Figure 9 shows only one band with a molecular weight of 88 kDa. This demonstrates that inulosucrase is successfully expressed and purified.

Figure 9: SDS-PAGE of inulosucrase from EC 2.4.1.9-containing E.coli BL21(DE3).
Figure 9: SDS-PAGE of inulosucrase from EC 2.4.1.9-containing E.coli BL21(DE3).

Other Test

We utilized thin-layer chromatography to verify that this enzyme can degrade sucrose. The principle of thin-layer chromatography is to use each component's different adsorption capacities on the same adsorbent so that the solvent flows through the adsorbent process, continuous adsorption, and desorption to achieve the mutual separation of the components. The reaction was carried out at 37°C for 1 hour in a buffer system with the addition of 2% plantain sugar and the appropriate amount of recombinant enzyme solution.

Figure 10: Results of thin-layer chromatography.
Figure 10: Results of thin-layer chromatography. 1: The recombinant enzyme solution with a ratio of 1:1 between dextransucrase and inulosucrase; 2: The recombinant enzyme solution with a ratio of 1:2 between dextransucrase and inulosucrase; 3: The recombinant enzyme solution with a ratio of 2:1 between dextransucrase and inulosucrase.

The results showed a reduction in sucrose and glucose production on the thin-layer chromatography plates when only 2% sucrose substrate was added, and the reaction was carried out at 37°C for 1 hour. This shows that this enzyme can break down sucrose to produce glucose and other substances in the reaction system.

In addition, sugarcane juice was used as a type of simulated food suspension to test enzyme function in real conditions. The experiment took place under 37°C to simulate human body temperature. A 1:1 ratio of concentration between dextransucrase and inulosucrase was used with masses of 1mg, 5mg, 10mg, 15mg, and 20mg of each type of enzyme per 100g sugarcane juice for 5 minutes, 10 minutes, 15 minutes, 30 minutes, and 60 minutes. Once the reaction time was reached, the solution was heated to 65°C to denature the enzymes.

Afterwards, HPLC was carried out to analyze the amount of sucrose and glucose in the solution. We used Origin Pro to process data, forming 3D mappings about glucose and sucrose concentrations in various conditions. By analyzing the raw data and the 3D mapping, we concluded that, in general, with a longer reaction time, catalyzing tends to become more complete, which means higher glucose concentration while lower sucrose concentration.

In speaking of each enzyme's sample size, except for the extremely high remaining sucrose with 1mg of dextransucrase and inulosucrase, it is difficult to summarize the overall effects of the change in glucose and sucrose concentrations. A low sucrose concentration (0g/100g) was left in the solution when 20mg of each enzyme was left and reacted for 60 minutes, but a higher glucose concentration was formed (2.9g/100g).

However, when the sample size is 10mg for each enzyme, noticeably low remaining sucrose (0.088g/100g) and relatively low glucose production (2.6g/100g) were observed when the reaction time was 15 minutes. In conclusion, we determined that allowing 10mg of each enzyme to react in sugarcane juice for 15 minutes is ideal to guarantee little sucrose sustain and promote glucose synthesis at 37°C.

Figure 11: 3D mapping of glucose and sucrose concentrations in various conditions under 37°C.
Figure 11: 3D mapping of glucose (right) and sucrose (left) concentrations in various conditions under 37°C.

References

  1. NCBI. Dextransucrase 2024. Accessed July 25, 2024.
  2. BRENDA. BRENDA:EC2.4.1.5 2023. Accessed June 6, 2024.
  3. Guan Z-W, Yu E-Z, Feng Q. Soluble Dietary Fiber, One of the Most Important Nutrients for the Gut Microbiota. Molecules 2021;26:6802. https://doi.org/10.3390/molecules26226802.
  4. Nie Y, Luo F. Dietary Fiber: An Opportunity for a Global Control of Hyperlipidemia. Oxid Med Cell Longev 2021;2021:5542342. https://doi.org/10.1155/2021/5542342.
  5. Te Morenga LA, Howatson AJ, Jones RM, Mann J. Dietary sugars and cardiometabolic risk: systematic review and meta-analyses of randomized controlled trials of the effects on blood pressure and lipids123. The American Journal of Clinical Nutrition 2014;100:65–79. https://doi.org/10.3945/ajcn.113.081521.
  6. Stanhope KL, Schwarz JM, Keim NL, Griffen SC, Bremer AA, Graham JL, et al. Consuming fructose-sweetened beverages, not glucose-sweetened ones, increases visceral adiposity and lipids and decreases insulin sensitivity in overweight/obese humans. J Clin Invest 2009;119:1322–34. https://doi.org/10.1172/JCI37385.
  7. Alexander Bentley R, Ruck DJ, Fouts HN. U.S. obesity as a delayed effect of excess sugar. Econ Hum Biol 2020;36:100818. https://doi.org/10.1016/j.ehb.2019.100818.
  8. Blüher M. Obesity: global epidemiology and pathogenesis. Nat Rev Endocrinol 2019;15:288–98. https://doi.org/10.1038/s41574-019-0176-8.
  9. Yu Z, Ley SH, Sun Q, Hu FB, Malik VS. Cross-sectional association between sugar-sweetened beverage intake and cardiometabolic biomarkers in US women. Br J Nutr 2018;119:570–80. https://doi.org/10.1017/S0007114517003841.
  10. Jebril M, Liu X, Shi Z, Mazidi M, Altaher A, Wang Y. Prevalence of Type 2 Diabetes and Its Association with Added Sugar Intake in Citizens and Refugees Aged 40 or Older in the Gaza Strip, Palestine. International Journal of Environmental Research and Public Health 2020;17:8594. https://doi.org/10.3390/ijerph17228594.
  11. Basu S, Yoffe P, Hills N, Lustig RH. The Relationship of Sugar to Population-Level Diabetes Prevalence: An Econometric Analysis of Repeated Cross-Sectional Data. PLOS ONE 2013;8:e57873. https://doi.org/10.1371/journal.pone.0057873.
  12. Maersk M, Belza A, Stødkilde-Jørgensen H, Ringgaard S, Chabanova E, Thomsen H, et al. Sucrose-sweetened beverages increase fat storage in the liver, muscle, and visceral fat depot: a 6-mo randomized intervention study. The American Journal of Clinical Nutrition 2012;95:283–9. https://doi.org/10.3945/ajcn.111.022533.
  13. Li Y, Hruby A, Bernstein AM, Ley SH, Wang DD, Chiuve SE, et al. Saturated Fats Compared With Unsaturated Fats and Sources of Carbohydrates about Risk of Coronary Heart Disease: A Prospective Cohort Study. J Am Coll Cardiol 2015;66:1538–48. https://doi.org/10.1016/j.jacc.2015.07.055.
  14. Pacheco LS, LaceyJr JV, Martinez ME, Lemus H, Araneta MRG, Sears DD, et al. Sugar‐Sweetened Beverage Intake and Cardiovascular Disease Risk in the California Teachers Study. Journal of the American Heart Association 2020. https://doi.org/10.1161/JAHA.119.014883.
  15. Saadatagah S, Pasha AK, Alhalabi L, Sandhyavenu H, Farwati M, Smith CY, et al. Coronary Heart Disease Risk Associated with Primary Isolated Hypertriglyceridemia; a Population-Based Study. J Am Heart Assoc 2021;10:e019343. https://doi.org/10.1161/JAHA.120.019343.
  16. Adeva-Andany MM, Martínez-Rodríguez J, González-Lucán M, Fernández-Fernández C, Castro-Quintela E. Insulin resistance is a cardiovascular risk factor in humans. Diabetes & Metabolic Syndrome: Clinical Research & Reviews 2019;13:1449–55. https://doi.org/10.1016/j.dsx.2019.02.023.
  17. Park WY, Yiannakou I, Petersen JM, Hoffmann U, Ma J, Long MT. Sugar-Sweetened Beverage, Diet Soda, and Nonalcoholic Fatty Liver Disease Over 6 Years: The Framingham Heart Study. Clinical Gastroenterology and Hepatology 2022;20:2524-2532.e2. https://doi.org/10.1016/j.cgh.2021.11.001.
  18. Janzi S, Ramne S, González-Padilla E, Johnson L, Sonestedt E. Associations Between Added Sugar Intake and Risk of Four Different Cardiovascular Diseases in a Swedish Population-Based Prospective Cohort Study. Front Nutr 2020;7. https://doi.org/10.3389/fnut.2020.603653.