Difference between revisions of "Part:BBa K5093002"
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<partinfo>BBa_K5093002 short</partinfo>
 
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short description
 
  
 
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===Functional Parameters===
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<html lang="en">
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<partinfo>BBa_K5093002 parameters</partinfo>
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<head>
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<!-- -->
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    <meta charset="UTF-8">
 +
    <meta name="viewport" content="width=device-width, initial-scale=1.0">
 +
    <title>EC 2.4.1.5 Gene Documentation</title>
 +
</head>
 +
<body>
 +
 
 +
    <!-- Gene Overview Section -->
 +
    <h2>Construction Design</h2>
 +
    <p>This engineered plasmid comprises a pET28a backbone (BBa_K3521004) and the gene EC 2.4.1.5 (BBa_K5093000), as shown in Figure 1.</p>
 +
 
 +
    <!-- Figure 1 -->
 +
    <div style="text-align:center;">
 +
        <img src="https://static.igem.wiki/teams/5093/bba-k5093000/1.png" width="50%" alt="Figure 1: Plasmid maps of pET28a-EC.2.4.1.5">
 +
        <div style="text-align:center;">
 +
            <caption>Figure 1: Plasmid maps of pET28a-EC.2.4.1.5</caption>
 +
        </div>
 +
    </div>
 +
 
 +
    <p>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.</p>
 +
 
 +
    <!-- Figure 2 -->
 +
    <div style="text-align:center;">
 +
        <img src="https://static.igem.wiki/teams/5093/bba-k5093001/2.png" width="50%" alt="Figure 2: The formation of dextran with dextransucrase from sucrose">
 +
        <div style="text-align:center;">
 +
            <caption>Figure 2: The formation of dextran with dextransucrase from sucrose [18]</caption>
 +
        </div>
 +
    </div>
 +
 
 +
    <h3>T7 Promoter (BBa_K3521002) and T7 Terminator (BBa_K3521002)</h3>
 +
    <p>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 IPTG is added, it binds to the Lac repressor, allowing T7 RNA polymerase to bind to the T7 promoter and initiate transcription.</p>
 +
 
 +
    <!-- Figure 3 -->
 +
    <div style="text-align:center;">
 +
        <img src="https://static.igem.wiki/teams/5093/bba-k5093001/3.png" width="50%" alt="Figure 3: An illustration of the T7 promoter and terminator">
 +
        <div style="text-align:center;">
 +
            <caption>Figure 3: An illustration of the T7 promoter and terminator</caption>
 +
        </div>
 +
    </div>
 +
 
 +
    <h3>Plasmid backbone: pET28a (BBa_K3521004)</h3>
 +
    <p>The pET28a plasmid has features like multiple restriction sites, a 6×histidine tag, and a kanamycin resistance gene. This enables efficient transformation and selection of successful clones.</p>
 +
 
 +
    <h3>Experimental Approach</h3>
 +
    <h4>1. Obtaining, Amplifying, and Identifying the Gene</h4>
 +
    <p>The gene EC 2.4.1.5 was synthesized by GeneScript and cut with NdeI and XhoI for ligation into pET28a. Successful amplification was confirmed by gel electrophoresis, shown in Figure 4.</p>
 +
 
 +
    <!-- Figure 4 -->
 +
    <div style="text-align:center;">
 +
        <img src="https://static.igem.wiki/teams/5093/bba-k5093001/4.jpg" width="50%" alt="Figure 4: The results of gel electrophoresis of EC 2.4.1.5">
 +
        <div style="text-align:center;">
 +
            <caption>Figure 4: The results of gel electrophoresis of EC 2.4.1.5</caption>
 +
        </div>
 +
    </div>
 +
 
 +
    <h4>2. Plasmid Construction and Transformation</h4>
 +
    <p>pET28a was cut with NdeI and XhoI, and EC 2.4.1.5 was ligated to it. Successful transformation into E. coli DH5α was confirmed by colony PCR and gel electrophoresis (Figure 5A), followed by plasmid sequencing (Figure 6).</p>
 +
 
 +
    <!-- Figure 5 -->
 +
    <div style="text-align:center;">
 +
        <img src="https://static.igem.wiki/teams/5093/bba-k5093001/5.jpg" width="50%" alt="Figure 5: A: Gel electrophoresis to confirm pET28a-EC 2.4.1.5 plasmids. B: pET28a-EC 2.4.1.5 containing strain clones">
 +
        <div style="text-align:center;">
 +
            <caption>Figure 5: A: Gel electrophoresis to confirm pET28a-EC 2.4.1.5 plasmids. B: pET28a-EC 2.4.1.5 containing strain clones</caption>
 +
        </div>
 +
    </div>
 +
 
 +
    <!-- Figure 6 -->
 +
    <div style="text-align:center;">
 +
        <img src="https://static.igem.wiki/teams/5093/bba-k5093000/6.png" width="50%" alt="Figure 6: The DNA sequencing diagram for pET28a-EC 2.4.1.5">
 +
        <div style="text-align:center;">
 +
            <caption>Figure 6: The DNA sequencing diagram for pET28a-EC 2.4.1.5</caption>
 +
        </div>
 +
    </div>
 +
 
 +
    <h4>3. Protein Expression</h4>
 +
    <p>The verified plasmids were transformed into E.coli BL21(DE3), and protein expression was induced with IPTG. The size of dextransucrase (167 kDa) was confirmed by SDS-PAGE (Figure 8).</p>
 +
 
 +
    <!-- Figure 7 -->
 +
    <div style="text-align:center;">
 +
        <img src="https://static.igem.wiki/teams/5093/bba-k5093000/7.jpg" width="50%" alt="Figure 7: DNA gel electrophoresis of EC 2.4.1.5 (A) and clones of E.coli BL21(DE3) containing the gene (B)">
 +
        <div style="text-align:center;">
 +
            <caption>Figure 7: DNA gel electrophoresis of EC 2.4.1.5 (A) and clones of E.coli BL21(DE3) containing the gene (B)</caption>
 +
        </div>
 +
    </div>
 +
 
 +
    <!-- Figure 8 -->
 +
    <div style="text-align:center;">
 +
        <img src="https://static.igem.wiki/teams/5093/bba-k5093000/8.jpg" width="50%" alt="Figure 8: SDS-PAGE of dextransucrase from EC 2.4.1.5-containing E.coli BL21(DE3)">
 +
        <div style="text-align:center;">
 +
            <caption>Figure 8: SDS-PAGE of dextransucrase from EC 2.4.1.5-containing E.coli BL21(DE3)</caption>
 +
        </div>
 +
    </div>
 +
 
 +
    <h4>4. Protein Purification</h4>
 +
    <p>Nickel affinity chromatography was used to purify dextransucrase. Figure 8 confirms successful purification with a clear band at 167 kDa.</p>
 +
 
 +
    <h4>Other Test</h4>
 +
    <p>Thin-layer chromatography verified the enzyme's ability to degrade sucrose. Reaction conditions and the effect of enzyme concentrations are shown in Figure 9.</p>
 +
 
 +
    <!-- Figure 9 -->
 +
    <div style="text-align:center;">
 +
        <img src="https://static.igem.wiki/teams/5093/bba-k5093000/9.png" width="50%" alt="Figure 9: Thin-layer chromatography results">
 +
        <div style="text-align:center;">
 +
            <caption>Figure 9: Thin-layer chromatography results</caption>
 +
        </div>
 +
    </div>
 +
 
 +
    <p>We also tested the enzyme function in sugarcane juice to simulate real conditions. Figure 10 shows the 3D mapping of glucose and sucrose concentrations under various conditions.</p>
 +
 
 +
    <!-- Figure 10 -->
 +
    <div style="text-align:center;">
 +
        <img src="https://static.igem.wiki/teams/5093/bba-k5093000/10.png" width="50%" alt="Figure 10: 3D mapping of glucose and sucrose concentrations in various conditions under 37°C">
 +
        <div style="text-align:center;">
 +
            <caption>Figure 10: 3D mapping of glucose (right) and sucrose (left) concentrations in various conditions under 37°C</caption>
 +
        </div>
 +
    </div>
 +
 
 +
    <!-- References Section -->
 +
    <h3>References</h3>
 +
    <p>[1] NCBI. Dextransucrase 2024. <a href="https://www.ncbi.nlm.nih.gov/protein/BAF96719.1</a> (accessed July 25, 2024).</p>
 +
    <p>[2] BRENDA. BRENDA:EC2.4.1.5 2023. <a href="https://www.brenda-enzymes.org/enzyme.php?ecno=2.4.1.5">https://www.brenda-enzymes.org/enzyme.php?ecno=2.4.1.5</a> (accessed June 6, 2024).</p>
 +
    <p>[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. <a href="https://doi.org/10.3390/molecules26226802">https://doi.org/10.3390/molecules26226802</a>.</p>
 +
    <p>[4] Nie Y, Luo F. Dietary Fiber: An Opportunity for a Global Control of Hyperlipidemia. Oxid Med Cell Longev 2021;2021:5542342. <a href="https://doi.org/10.1155/2021/5542342">https://doi.org/10.1155/2021/5542342</a>.</p>
 +
    <p>[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. <a href="https://doi.org/10.3945/ajcn.113.081521">https://doi.org/10.3945/ajcn.113.081521</a>.</p>
 +
    <p>[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. <a href="https://doi.org/10.1172/JCI37385">https://doi.org/10.1172/JCI37385</a>.</p>
 +
    <p>[7] Alexander Bentley R, Ruck DJ, Fouts HN. U.S. obesity as a delayed effect of excess sugar. Econ Hum Biol 2020;36:100818. <a href="https://doi.org/10.1016/j.ehb.2019.100818">https://doi.org/10.1016/j.ehb.2019.100818</a>.</p>
 +
    <p>[8] Blüher M. Obesity: global epidemiology and pathogenesis. Nat Rev Endocrinol 2019;15:288–98. <a href="https://doi.org/10.1038/s41574-019-0176-8">https://doi.org/10.1038/s41574-019-0176-8</a>.</p>
 +
    <p>[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. <a href="https://doi.org/10.1017/S0007114517003841">https://doi.org/10.1017/S0007114517003841</a>.</p>
 +
    <p>[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. <a href="https://doi.org/10.3390/ijerph17228594">https://doi.org/10.3390/ijerph17228594</a>.</p>
 +
    <p>[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. <a href="https://doi.org/10.1371/journal.pone.0057873">https://doi.org/10.1371/journal.pone.0057873</a>.</p>
 +
    <p>[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. <a href="https://doi.org/10.3945/ajcn.111.022533">https://doi.org/10.3945/ajcn.111.022533</a>.</p>
 +
    <p>[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. <a href="https://doi.org/10.1016/j.jacc.2015.07.055">https://doi.org/10.1016/j.jacc.2015.07.055</a>.</p>
 +
    <p>[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. <a href="https://doi.org/10.1161/JAHA.119.014883">https://doi.org/10.1161/JAHA.119.014883</a>.</p>
 +
    <p>[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. <a href="https://doi.org/10.1161/JAHA.120.019343">https://doi.org/10.1161/JAHA.120.019343</a>.</p>
 +
    <p>[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. <a href="https://doi.org/10.1016/j.dsx.2019.02.023">https://doi.org/10.1016/j.dsx.2019.02.023</a>.</p>
 +
    <p>[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. <a href="https://doi.org/10.1016/j.cgh.2021.11.001">https://doi.org/10.1016/j.cgh.2021.11.001</a>.</p>
 +
    <p>[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. <a href="https://doi.org/10.3389/fnut.2020.603653">https://doi.org/10.3389/fnut.2020.603653</a>.</p>
 +
 
 +
</body>
 +
</html>

Revision as of 08:12, 28 September 2024

pET28a-EC.2.4.1.5


Usage and Biology

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 5302
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 4402
    Illegal BglII site found at 8423
    Illegal BamHI site found at 5915
  • 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 6571
    Illegal AgeI site found at 5125
    Illegal AgeI site found at 6837
    Illegal AgeI site found at 7882
    Illegal AgeI site found at 8032
    Illegal AgeI site found at 8272
    Illegal AgeI site found at 8383
    Illegal AgeI site found at 8443
  • 1000
    COMPATIBLE WITH RFC[1000]


<!DOCTYPE html> EC 2.4.1.5 Gene Documentation

Construction Design

This engineered plasmid comprises a pET28a backbone (BBa_K3521004) and the gene EC 2.4.1.5 (BBa_K5093000), as shown in Figure 1.

Figure 1: Plasmid maps of pET28a-EC.2.4.1.5
Figure 1: Plasmid maps of pET28a-EC.2.4.1.5

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 [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 IPTG is added, it binds to the Lac repressor, allowing T7 RNA polymerase to bind to the T7 promoter and initiate transcription.

Figure 3: An illustration of the T7 promoter and terminator
Figure 3: An illustration of the T7 promoter and terminator

Plasmid backbone: pET28a (BBa_K3521004)

The pET28a plasmid has features like multiple restriction sites, a 6×histidine tag, and a kanamycin resistance gene. This enables efficient transformation and selection of successful clones.

Experimental Approach

1. Obtaining, Amplifying, and Identifying the Gene

The gene EC 2.4.1.5 was synthesized by GeneScript and cut with NdeI and XhoI for ligation into pET28a. Successful amplification was confirmed by gel electrophoresis, shown in Figure 4.

Figure 4: The results of gel electrophoresis of EC 2.4.1.5
Figure 4: The results of gel electrophoresis of EC 2.4.1.5

2. Plasmid Construction and Transformation

pET28a was cut with NdeI and XhoI, and EC 2.4.1.5 was ligated to it. Successful transformation into E. coli DH5α was confirmed by colony PCR and gel electrophoresis (Figure 5A), followed by plasmid sequencing (Figure 6).

Figure 5: A: Gel electrophoresis to confirm pET28a-EC 2.4.1.5 plasmids. B: pET28a-EC 2.4.1.5 containing strain clones
Figure 5: A: Gel electrophoresis to confirm pET28a-EC 2.4.1.5 plasmids. B: pET28a-EC 2.4.1.5 containing strain clones
Figure 6: The DNA sequencing diagram for pET28a-EC 2.4.1.5
Figure 6: The DNA sequencing diagram for pET28a-EC 2.4.1.5

3. Protein Expression

The verified plasmids were transformed into E.coli BL21(DE3), and protein expression was induced with IPTG. The size of dextransucrase (167 kDa) was confirmed by SDS-PAGE (Figure 8).

Figure 7: DNA gel electrophoresis of EC 2.4.1.5 (A) and clones of E.coli BL21(DE3) containing the gene (B)
Figure 7: DNA gel electrophoresis of EC 2.4.1.5 (A) and clones of E.coli BL21(DE3) containing the gene (B)
Figure 8: SDS-PAGE of dextransucrase from EC 2.4.1.5-containing E.coli BL21(DE3)
Figure 8: SDS-PAGE of dextransucrase from EC 2.4.1.5-containing E.coli BL21(DE3)

4. Protein Purification

Nickel affinity chromatography was used to purify dextransucrase. Figure 8 confirms successful purification with a clear band at 167 kDa.

Other Test

Thin-layer chromatography verified the enzyme's ability to degrade sucrose. Reaction conditions and the effect of enzyme concentrations are shown in Figure 9.

Figure 9: Thin-layer chromatography results
Figure 9: Thin-layer chromatography results

We also tested the enzyme function in sugarcane juice to simulate real conditions. Figure 10 shows the 3D mapping of glucose and sucrose concentrations under various conditions.

Figure 10: 3D mapping of glucose and sucrose concentrations in various conditions under 37°C
Figure 10: 3D mapping of glucose (right) and sucrose (left) concentrations in various conditions under 37°C

References

[1] NCBI. Dextransucrase 2024. 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.