Difference between revisions of "Part:BBa K5083000"

 
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Glucagon-like peptide-1 (GLP-1) is a hormone primarily produced by intestinal L cells and belongs to the incretin family. GLP-1 functions include promoting insulin secretion, reducing glucagon secretion, inhibiting gastric emptying, and decreasing gastric acid secretion. GLP-1 can be used to treat type 2 diabetes and obesity. It is one of the incretin hormones, derived from the cleavage of proglucagon. Proglucagon is a peptide hormone composed of 160 amino acids and is found in intestinal L cells or the brain. After being cleaved by prohormone convertase 1 (PC1), it is split into two different forms of GLP-1: GLP-1 (7–36) amide, a 30-amino acid amidated form, and GLP-1 (7–37), a 31-amino acid form extended by glycine. The majority of biologically active GLP-1 in the human body exists as GLP-1 (7–36) amide. We demonstrated through ELISA and Western blotting that GLP-1 can be expressed in Escherichia coli BL21 and Nissle 1917.
  
 
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===Usage and Biology===
 
===Usage and Biology===
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Description
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Given that obesity can lead to a range of health problems and current weight loss methods are often inefficient, time-consuming, or expensive, we are developing a probiotic that can express GLP-1. GLP-1, which originates from intestinal L cells, has been shown to promote insulin secretion, reduce glucagon secretion, inhibit gastric emptying, and decrease gastric acid secretion.
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Usage and Biology
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We utilized the ribosome binding site from the iGEM part library (BBa_B0034) followed by the GLP-1 sequence fragment. To express GLP-1, we employed a constitutive promoter (BBa_J23100) and a strong terminator (BBa_B0015). Finally, we used the pET23b vector to construct the plasmid and transformed it into Escherichia coli BL21.
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<img src="https://static.igem.wiki/teams/5083/1.png" style="width: 500px;margin: 0 auto" />
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<p style="font-size: 98%; line-height: 1.4em;">Fig 1.genetic circuit diagram in BL21</p >
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==Characterization==
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We successfully amplified the GLP-1 fragment and expressed it in both Escherichia coli BL21 and Nissle 1917.
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<img src="https://static.igem.wiki/teams/5083/7.png" style="width: 500px;margin: 0 auto" />
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<p style="font-size: 98%; line-height: 1.4em;">Fig 2.Fragment amplification validation.</p >
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<img src="https://static.igem.wiki/teams/5083/2.jpg" style="width: 500px;margin: 0 auto" />
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<p style="font-size: 98%; line-height: 1.4em;">Fig 3.GLP-1 expression in BL21</p >
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<img src="https://static.igem.wiki/teams/5083/3.png" style="width: 500px;margin: 0 auto" />
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<p style="font-size: 98%; line-height: 1.4em;">Fig 4.Western blotting experiment validation.</p >
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As shown in the figure, the engineered strains can effectively express GLP-1 in vivo, reaching a concentration of 59.8 pg/mL.
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<img src="https://static.igem.wiki/teams/5083/4.png" style="width: 500px;margin: 0 auto" />
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<p style="font-size: 98%; line-height: 1.4em;">Fig 5.genetic circuit diagram in EcN</p >
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<img src="https://static.igem.wiki/teams/5083/5.png" style="width: 500px;margin: 0 auto" />
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<p style="font-size: 98%; line-height: 1.4em;">Fig 6.ELISA validation of GLP-1 expression.</p >
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Finally, we constructed an engineered plasmid suitable for Escherichia coli Nissle 1917 and expressed GLP-1 in EcN. Ultimately, using an ELISA kit, we detected an extracellular GLP-1 concentration of 33.1 pg/mL.
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Potential application directions
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This experiment demonstrates that human-derived GLP-1 can be heterologously expressed in Escherichia coli. This opens up possibilities for developing probiotics for diabetes treatment or weight loss. It holds promise for addressing the high costs associated with the complex production processes and low yields of current GLP-1 production methods, offering significant potential for future development.
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References
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[1] Li Y, Hansotia T, Yusta B, et al. Glucagon-like peptide-1 receptor signaling modulates β cell apoptosis[J]. Journal of Biological Chemistry, 2003, 278(1): 471-478.
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[2] Kim K S, Park J S, Hwang E, et al. GLP-1 increases preingestive satiation via hypothalamic circuits in mice and humans[J]. Science, 2024, 385(6707): 438-446.
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[3] Kobayati A, Haidar A, Tsoukas M A. Glucagon‐like peptide‐1 receptor agonists as adjunctive treatment for type 1 diabetes: Renewed opportunities through tailored approaches?[J]. Diabetes, Obesity and Metabolism, 2022, 24(5): 769-787.
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Revision as of 01:48, 14 September 2024


GLP-1

Glucagon-like peptide-1 (GLP-1) is a hormone primarily produced by intestinal L cells and belongs to the incretin family. GLP-1 functions include promoting insulin secretion, reducing glucagon secretion, inhibiting gastric emptying, and decreasing gastric acid secretion. GLP-1 can be used to treat type 2 diabetes and obesity. It is one of the incretin hormones, derived from the cleavage of proglucagon. Proglucagon is a peptide hormone composed of 160 amino acids and is found in intestinal L cells or the brain. After being cleaved by prohormone convertase 1 (PC1), it is split into two different forms of GLP-1: GLP-1 (7–36) amide, a 30-amino acid amidated form, and GLP-1 (7–37), a 31-amino acid form extended by glycine. The majority of biologically active GLP-1 in the human body exists as GLP-1 (7–36) amide. We demonstrated through ELISA and Western blotting that GLP-1 can be expressed in Escherichia coli BL21 and Nissle 1917.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]