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             <caption>Figure 2: The formation of dextran with dextransucrase from sucrose [2]</caption>
 
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             <caption>Figure 3: The results of gel electrophoresis of EC 2.4.1.5</caption>
 
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         <img src="https://static.igem.wiki/teams/5093/bba-k5189007/4.jpg" width="50%" alt="Figure 4: SDS-PAGE of dextransucrase from EC 2.4.1.5-containing E.coli BL21(DE3)">
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         <img src="https://static.igem.wiki/teams/5093/bba-k5093000/4.jpg" width="30%" alt="Figure 4: SDS-PAGE of dextransucrase from EC 2.4.1.5-containing E.coli BL21(DE3)">
 
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             <caption>Figure 4: SDS-PAGE of dextransucrase from EC 2.4.1.5-containing E.coli BL21(DE3)</caption>
 
             <caption>Figure 4: SDS-PAGE of dextransucrase from EC 2.4.1.5-containing E.coli BL21(DE3)</caption>

Latest revision as of 05:20, 29 September 2024

EC.2.4.1.5

short description


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 883
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 4004
    Illegal BamHI site found at 1496
    Illegal XhoI site found at 4435
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


EC 2.4.1.5 Gene Documentation

EC 2.4.1.5 Gene

Base Pairs: 4440bp
Origin: Leuconostoc citreum [1], synthesized

Properties

EC 2.4.1.5 codes for the enzyme dextransucrase, which transfers a D-glucosyl group from sucrose, a disaccharide, to a growing dextran chain, a fiber, by an alpha (1→6) linkage. The reaction is illustrated in Figure 2.

Figure 1: Gene map of EC 2.4.1.5
Figure 1: Gene map of EC 2.4.1.5

This new essential part, EC 2.4.1.5, originated in Leuconostoc citreum [1] and is 4440 bp long, as shown in Figure 1. The biotech company Gene Script artificially synthesized the gene samples we used. EC 2.4.1.5 codes for the enzyme dextransucrase, which transfers a D-glucosyl group from sucrose, a disaccharide, to a growing dextran chain, a fiber, by an alpha (1→6) linkage. The reaction is illustrated in Figure 2.

Figure 2: The formation of dextran with dextransucrase from sucrose
Figure 2: The formation of dextran with dextransucrase from sucrose [2]

Use of Dextransucrase

Sucrose is usually broken down into two monosaccharides, glucose and fructose, which are absorbed and metabolized. However, the protein that the gene codes for converts this disaccharide into 50% fructose and 50% dextran. This is the primary approach to dextran synthesis.

Dextran is a versatile substance. As a soluble dietary fiber, it can be efficiently fermented by the gut microbiota. 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 [3]. Furthermore, dietary fiber could lower blood lipid concentration, reducing the risk of hyperlipidemia [4].

Its medical uses include its antithrombotic and anticoagulant activities and its role in maintaining blood osmolarity to replace expensive plasma proteins. Its excellent biological compatibility and degradability have made it a promising drug administration vector. Dextran is also a food additive with great stabilizing, thickening, and emulsifying abilities. In cosmetics, dextran and its derivatives also function as lubricants, humectants, and thickeners in skin and hair care products. Dextran could also facilitate imaging technologies and the manufacture of paper.

The majority of attention on dextransucrase is associated with the functions of dextran. However, as our project aims to synthesize dextran in the human gut, its effect on its substrate, sucrase, is also vital. It reduces the absorption of glucose by adding them to dextran chains, and hence 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 [5–18], without diminishing the sweetness of food and drink.

Obtaining, Amplifying, and Identifying the Gene

The biotechnology company Gene Script 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 4440 bp. The bands representing EC 2.4.1.5 successfully appear at their corresponding positions in the gel, as shown in Figure 3, indicating that the cutting and amplifying are successful.

Figure 3: The results of gel electrophoresis of EC 2.4.1.5
Figure 3: The results of gel electrophoresis of EC 2.4.1.5

Protein Purification and SDS-PAGE

Nickel affinity chromatography effectively purifies dextransucrase because the protein contains a 6×his tag. We got a more precise result with little interference by non-specifically bound proteins. Figure 4 shows only one band with a molecular weight of 167 kDa. This demonstrates that dextransucrase is successfully expressed and purified.

Figure 4: SDS-PAGE of dextransucrase from EC 2.4.1.5-containing E.coli BL21(DE3)
Figure 4: SDS-PAGE of dextransucrase from EC 2.4.1.5-containing E.coli BL21(DE3)

References

[1] NCBI. Dextransucrase 2024. https://www.ncbi.nlm.nih.gov/protein/BAF96719.1 (accessed July 25, 2024).

[2] BRENDA. BRENDA:EC2.4.1.5 2023. https://www.brenda-enzymes.org/enzyme.php?ecno=2.4.1.5 (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 lipids. 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.