Part:BBa_K5023013

PAR_Ble_SP7_FAST-PETase_mCherry_Ter

This composite part have: PAR promoter, the bleomycin/zeocin resistance sequence, signal peptide, the Fast-PETase, gene reporter mCherry, and the terminator. Designed to express and secrete Fast-PETase with verification by mCherry reporter in Chlamydomonas reinhardtii, utilizing pJP32 backbone.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 698
  Illegal PstI site found at 2111
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 249
  Illegal NheI site found at 1211
  Illegal PstI site found at 698
  Illegal PstI site found at 2111
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1616
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 698
  Illegal PstI site found at 2111
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 698
  Illegal PstI site found at 2111
  Illegal NgoMIV site found at 1079
  Illegal NgoMIV site found at 1140
  Illegal NgoMIV site found at 1420
  Illegal NgoMIV site found at 3174
  
• 1000
  COMPATIBLE WITH RFC[1000]

Introduction to the Mhetygua Project

The Mhetygua project, initiated for the IGEM Global 2023 edition, represents a pioneering endeavor to combat the pressing issue of microplastic pollution. Spearheaded by a dedicated group of students, the project titled "MHETYGUÁ: algae as devices for the degradation of aquatic pollutants" seeks to harness the potential of algae, specifically Chlamydomonas reinhardtii, as a bio-tool for microplastic degradation. Beyond the scientific pursuit, the project embodies a broader vision of fostering environmental awareness and education, aiming to catalyze a shift towards sustainable practices and a reduced plastic footprint in the Latin American region.

Commentary on the Genetic Circuit Construction: Circuit Number 3

The genetic circuit, labeled as Circuit Number 3, is meticulously designed to achieve the expression and secretion of the enzyme Fast-PETase in the microalgae Chlamydomonas reinhardtii, with the added feature of verification through the mCherry reporter gene. The construction of this circuit involves several key components:

1. PAR Promoter: Serving as the starting point of the circuit, the PAR promoter is chosen for its light-inducible properties, allowing for the modulation of gene expression based on light availability. This promoter is known for its efficacy in Chlamydomonas reinhardtii, making it an ideal choice for initiating the transcription of downstream genes.
2. Bleomycin/Zeocin Resistance Sequence: This sequence confers resistance to the antibiotics bleomycin and zeocin. By incorporating this resistance gene, it becomes feasible to select for algal cells that have successfully integrated the genetic circuit, ensuring the propagation of the desired trait.
3. Signal Peptide: A crucial component for ensuring the secretion of the enzyme, the signal peptide directs the synthesized Fast-PETase protein to the secretory pathway, facilitating its release into the external environment.
4. Fast-PETase: The central player of the circuit, this enzyme is engineered for enhanced plastic degradation capabilities. Once expressed and secreted, this enzyme acts on microplastics, breaking them down into less harmful byproducts.
5. Gene Reporter mCherry: mCherry is a fluorescent protein that emits a bright red fluorescence. Its inclusion in the circuit serves as a reporter gene, allowing researchers to visually verify the successful expression of Fast-PETase in the algal cells. The presence of red fluorescence indicates the active expression of the circuit.
6. Terminator: Concluding the circuit, the terminator sequence ensures the proper end of transcription, providing stability and efficiency to the overall genetic expression process.

The entire circuit is integrated into the pJP32 backbone, a vector known for its compatibility with Chlamydomonas reinhardtii. This choice of backbone further ensures the successful integration and expression of the circuit within the algal cells.