Part:BBa_K5292405

ICCG-GFP

ICCG is a mutant derived from LCC (leaf-branch compost cutinase), an enzyme originating from microorganisms capable of degrading plastic. The ICCG variant was developed through mutations and optimizations of the LCC sequence to enhance its plastic-degrading abilities. GFP (Green Fluorescent Protein) originates from the jellyfish Aequorea victoria, and it is used in the fusion protein as a fluorescent marker. The GSlinker is an artificially designed flexible linker peptide that connects ICCG and GFP while maintaining the functional independence of both proteins.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 72
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 862
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 68
  Illegal AgeI site found at 220
  Illegal AgeI site found at 621
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1555
  

Abstract

This signal peptide was selected through model prediction from a library of approximately 200 signal peptides in Escherichia coli, aiming to enhance the secretion expression of the PET-degrading enzyme ICCG. This signal peptide originates from... and was initially used to express... We constructed the signal peptide at the N-terminus of the ICCG-GFP fusion protein and characterized its secretion efficiency through fluorescence intensity. Although the secretion efficiency of this signal peptide in the experiment was not significantly improved, it provided valuable reference data for further optimization of PET-degrading enzyme expression.

In projects, this signal peptide can be used to guide the secretion expression of target proteins, especially in studies of recombinant protein secretion and applications in synthetic biology. Combining model predictions and experimental data, this signal peptide lays the foundation for subsequent optimization of signal peptides.

Profile

Name：ICCG-GFP
Base Pairs:1527
Origin:ICCG is a mutant derived from LCC (leaf-branch compost cutinase), an enzyme originating from microorganisms capable of degrading plastic. The ICCG variant was developed through mutations and optimizations of the LCC sequence to enhance its plastic-degrading abilities. GFP (Green Fluorescent Protein) originates from the jellyfish Aequorea victoria, and it is used in the fusion protein as a fluorescent marker. The GSlinker is an artificially designed flexible linker peptide that connects ICCG and GFP while maintaining the functional independence of both proteins
Properties: The ICCG-GFP fusion protein combines the plastic-degrading activity of ICCG with the fluorescence properties of GFP. GFP is linked to the C-terminus of ICCG using a GSlinker, ensuring both proteins maintain their independent functions without interference. The DMtac promoter and MnfaA signal peptide enhance protein expression and secretion, ensuring efficient expression and functional display in *E. coli*. The fluorescence intensity of GFP allows rapid quantification of ICCG expression.

Usage and Biology

The ICCG-GFP fusion protein is primarily used for the rapid quantification of ICCG expression levels in E. coli. By measuring the fluorescence intensity of GFP, it is possible to easily monitor the expression and secretion efficiency of ICCG, making it ideal for screening the regulatory effects of different promoters and signal peptides. The high-throughput screening system accelerates the optimization of promoters (e.g., DMtac, Mtac) and signal peptides (e.g., MnfaA), suitable for synthetic biology projects requiring precise gene expression regulation.

Characterization

We used seamless cloning to construct the GFP protein at the C-terminus of ICCG using the GS linker.
- Through shake flask cultivation and protein purification, we obtained the ICCG-GFP fusion protein sample. The results above indicate that we successfully constructed the correct ICCG-GFP fusion protein.The above results indicate that we successfully constructed the correct ICCG-GFP fusion protein.
- After purifying the ICCG-GFP fusion protein, we measured the fluorescence intensity of protein samples at final concentrations of 0 μg/mL, 2 μg/mL, 4 μg/mL, 8 μg/mL, 16 μg/mL, 32 μg/mL, and 64 μg/mL under excitation wavelength of 488 nm and emission wavelength of 507 nm, resulting in a fluorescence intensity versus protein concentration standard curve

Figure 1. PCR amplification results. According to the agarose gel electrophoresis results, PCR amplification yielded fragments of the expected length.

Figure 2. SDS-PAGE results of protein purification products.

Figure 3. Fluorescence intensity - protein concentration standard curve.

Reference

Tournier V, Topham CM, Gilles A, David B, Folgoas C, Moya-Leclair E, Kamionka E, Desrousseaux ML, Texier H, Gavalda S, Cot M, Guémard E, Dalibey M, Nomme J, Cioci G, Barbe S, Chateau M, André I, Duquesne S, Marty A. An engineered PET depolymerase to break down and recycle plastic bottles. Nature. 2020 Apr;580(7802):216-219. doi: 10.1038/s41586-020-2149-4. Epub 2020 Apr 8. PMID: 32269349.