Composite

Part:BBa_K5283022

Designed by: sherryqin   Group: iGEM24_AFMU-China   (2024-09-27)
Revision as of 14:31, 1 October 2024 by Radium (Talk | contribs) (→‎Plasmid Construction)


pGroESL-SPusp45-LEISSTCDA-Linker-LMWP-FGF21-histag Plasmid Detailed Description1. GroESL Inducible Promoter Function: The GroESL promoter is regulated by heat shock proteins and is typically activated under stress conditions, such as heat shock or chemical induction. Source: Derived from Escherichia coli heat shock protein genes, commonly utilized in industrial microbiology or synthetic biology. Application: Its inducible nature makes it suitable for controlled protein expression, particularly when expression needs to be initiated under specific conditions, aiding in maximizing protein yield and stability. 2. SPusp45 Signal Peptide Function: SPusp45 is a signal peptide sequence that directs the secretion of proteins out of the cell. Source: Typically sourced from the secretion system of Lactococcus lactis. Applicatio*: Used for secretory expression of recombinant proteins, enhancing solubility and purity, facilitating subsequent purification and application. 3. LEISSTCDA Sequence Function: LEISSTCDA is a peptide sequence used for protein modification or tagging. Source: This sequence may be obtained through in vitro selection or design, with specific functions depending on experimental requirements. Application: Employed to enhance protein functionality or facilitate detection and purification. 4. Linker (Peptide Linker) Function: Peptide linkers provide flexibility between protein domains, preventing structural interference. Source: Common linker sequences include (Gly_4Ser)_n, typically obtained through design. Application: Used to connect different functional domains, ensuring each domain can correctly fold and function independently. 5. LMWP (Low Molecular Weight Poly-Lysine) Function: LMWP is a cell-penetrating peptide sequence that aids in translocating proteins across cell membranes. Source: Generally, it is artificially designed or selected through screening. Application: Used in drug delivery systems to enhance intracellular delivery efficiency of genes or drugs. 6. FGF21 (Fibroblast Growth Factor 21) Function: FGF21 is a metabolic regulatory protein with roles in regulating glucose and lipid metabolism.**Source**: Naturally occurring in the human body and produced via gene cloning and expression systems. Application: Important in the research and treatment of metabolic diseases such as diabetes and obesity, potentially serving as a therapeutic protein drug. 7. His Tag Function: The His tag is a sequence of six histidines used for protein purification. Source: Widely utilized in molecular biology tools, added to recombinant proteins via genetic engineering. Application: Facilitates purification using nickel affinity chromatography (Ni-NTA), enabling high-purity target protein recovery. Potential Applications1. Metabolic Research: By inducing FGF21 expression, researchers can investigate its mechanisms in metabolic diseases.2. Protein Drug Development: Production and purification of FGF21 for preclinical research and drug development for metabolic disorders.3. Gene Therapy: Utilizing the cell-penetrating properties of LMWP to explore the feasibility of intracellular delivery of FGF21 genes or proteins.4. Industrial Production: Using the GroESL inducible promoter to mass-produce FGF21 in industrial microorganisms, reducing production costs Summary This plasmid combines multiple functional sequences to efficiently express and secrete FGF21 protein under specific conditions, facilitating subsequent purification and application. It holds significant potential for research and preclinical development in various fields.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 338
    Illegal suffix found in sequence at 948
    Illegal PstI site found at 147
    Illegal PstI site found at 535
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 338
    Illegal SpeI site found at 949
    Illegal PstI site found at 147
    Illegal PstI site found at 535
    Illegal PstI site found at 963
    Illegal NotI site found at 344
    Illegal NotI site found at 956
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 338
    Illegal BglII site found at 927
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 338
    Illegal suffix found in sequence at 949
    Illegal PstI site found at 147
    Illegal PstI site found at 535
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 338
    Illegal XbaI site found at 353
    Illegal SpeI site found at 949
    Illegal PstI site found at 147
    Illegal PstI site found at 535
    Illegal PstI site found at 963
  • 1000
    COMPATIBLE WITH RFC[1000]



Plasmid Construction

In the construction of engineered *L. lactis strain NZ9000* for therapy for diabetes, FGF21 is our key functional molecule. To achieve a long-lasting and non-invasive drug delivery method for diabetes,Due to the rapid onset of action of FGF21 and the wide range of receptor cells, we designed a cholate-induced secretion of FGF21. However, FGF21 needs to enter the circulatory system to function. The intestinal epithelium, composed of tightly connected intestinal columnar cells, restricts the passage of typical large molecular proteins. Additionally, the reliable, safe, and efficient delivery of proteins in the intestinal tract is challenging due to various factors, such as intestinal proteases(e.g., pancreatic proteases) and pH. By integrating FGF21 and LMWP (low molecular weight protamine) (BBa_K5283017) , which mediates the endocytosis of intestinal epithelial cells, we not only enhance the overall stability of the fusion protein but also ensure a safer and more reliable targeted cellular uptake compared to previous "intestinal penetration" strategies that compromised the integrity of the intestinal mucosal barrier. Regarding the LMWP connection issue, in order not to affect the binding of FGF21 to its receptor, we found by modeling molecular docking that LMWP attachment to the N-terminus of FGF21 did not affect its biological activity (plus linkage to the model), so we constructed LMWP-FGF21 fusion protein. In order to improve the secretion efficiency of lactic acid bacteria, we also designed an enhancer peptide LEISSTCDA (BBa_K5283014) matched with promoter USP45.

Figure 1. Changes in postprandial cholate in normal people.

Figure 2. The design of FGF21 plasmid.

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