Coding

Part:BBa_K5327013

Designed by: Yifan Zhang   Group: iGEM24_BUCT   (2024-08-28)


Cytochrome b5

Function:[1][2]

Membrane bound hemoprotein which function as an electron carrier for several membrane bound oxygenases. It plays a role in fatty-acid desaturation and is also involved in several steps of the sterol biosynthesis pathway, particularly in the 4-demethylation of the 4,4'-dimethyl zymosterol.

Usage and Biology

Genome localization:Chromosome XIV - NC_001146.8

Expression diagram:

Fig 1. The expression diagram of cytochrome b5

Corresponding enzyme structure:

Fig 2. The corresponding enzyme structure of cytochrome b5

The PCR result:

Fig 3. The PCR result of cytochrome b5

Subcellular localization:[3]

Located in the endoplasmic reticulum membrane and microsome membrane of cells

Fig 4. The subcellular localization of cytochrome b5

Dynamics data:

Table 1. The dynamics data of cytochrome b5

Design Notes

The Cytochrome b5 gene was designed based on the coding sequence (CDS) from Arabidopsis thaliana and was codon-optimized for Saccharomyces cerevisiae (S288C) to ensure efficient expression in yeast. Cytochrome b5 is a membrane-bound hemoprotein that acts as an electron carrier for various membrane-bound oxygenases, playing significant roles in fatty acid desaturation and sterol biosynthesis, particularly in the 4-demethylation of 4,4'-dimethyl zymosterol. In this experiment, Cytochrome b5 mainly functions as an auxiliary electron transfer protein, supporting the targeted metabolic reactions. The gene is expressed using the GPD promoter (GPDpBBa_K517001) and PYK1 terminator (PYK1tBBa_K5327018) to ensure high enzyme expression and mRNA stability in yeast. The optimized gene is inserted into a vector and introduced into S. cerevisiae S288C through homologous recombination, followed by screening and expression verification using deficient strains. This design aims to enhance the expression level of Cytochrome b5 in yeast, boosting its role in fatty acid metabolism and sterol biosynthesis pathways, thereby optimizing yeast as a metabolic engineering platform.

Plasmid

Fig 5. The plasmid expression of cytochrome b5

Source

Arabidopsis thaliana


References

  1. ↑ MAUK A G, MAUK M R, MOORE G R, et al. Experimental and theoretical analysis of the interaction between cytochrome c and cytochrome b5 [J]. Journal of bioenergetics and biomembranes, 1995, 27(3): 311-30.
  2. ↑ MüLLEDER M, CALVANI E, ALAM M T, et al. Functional Metabolomics Describes the Yeast Biosynthetic Regulome [J]. Cell, 2016, 167(2): 553-65.e12.
  3. ↑ https://www.uniprot.org/uniprotkb/P40312/entry


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]


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Parameters
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