Part:BBa_K5327023:Design

NAD(P) transhydrogenase subunit alpha

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 628
  Illegal EcoRI site found at 1267
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 628
  Illegal EcoRI site found at 1267
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 628
  Illegal EcoRI site found at 1267
  Illegal XhoI site found at 634
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 628
  Illegal EcoRI site found at 1267
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 628
  Illegal EcoRI site found at 1267
  Illegal NgoMIV site found at 1104
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

The design of the NAD(P) transhydrogenase subunit alpha gene is based on the known coding sequence from Saccharomyces cerevisiae and has undergone codon optimization for efficient expression in yeast. This enzyme catalyzes the transhydrogenation between NADH and NADP, coupling this reaction with respiration and ATP hydrolysis while functioning as a proton pump across the membrane. The gene is expressed using the ADH1 promoter (ADH1p BBa_J63005) and the TDH2 terminator (TDH2t BBa_K2407108) to ensure high levels of expression and mRNA stability in yeast. The optimized gene is inserted into an expression vector and introduced into the Saccharomyces cerevisiae S288C strain via homologous recombination, with screening and expression validation conducted using a defective strain. This design aims to enhance the activity of NAD(P) transhydrogenase in yeast, thereby improving cellular energy metabolism efficiency and providing a more effective functional platform for metabolic engineering.

Plasmid

Fig 1. The plasmid expression of NAD(P) transhydrogenase subunit alpha

Source

Arabidopsis thaliana