Part:BBa_K4347011:Design

Bst fusion with Sac7e and point mutations for enhanced thermal stability codon optimized for E.coli

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 5
  Illegal XhoI site found at 209
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1015
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

This fusion polymerase was the final iteration of the re-engineered Bst, incorporating an improved thermally stable version of Bst and thermal stable DNA binding protien Sac7e. An AlphaFold analysis in combination with Pymol was used to predict the structure of this synthetic polymerase. Sac7e was fused via N-terminal to Bst using a flexible (GGGGS)₄ linker.

Fully modified Bst polymerase with fusion protien and point mutations modelled in Pymol.

Bst Mutagenesis

For more information and a complete overview on how the mutated polymerase was designed please view: https://parts.igem.org/Part:BBa_K4347007:Design

DNA Binding Protien

For more information on why Sac7e was selected please view the considerations section on:https://parts.igem.org/Part:BBa_K4347010:Design#Considerations

Considerations

Point mutations were made for thermal stability to account for fluctuations in the portable heating device, as temperature fluctuations typically oscillate about the desired set point temperature when using electronic circuits.

Section can be summary from https://parts.igem.org/Part:BBa_K4347007# and https://parts.igem.org/Part:BBa_K4347010.

Source

• Sac7e: https://parts.igem.org/Part:BBa_K4347006
• (GGGGS)₄ linker: https://parts.igem.org/Part:BBa_K3117004
• Bst: https://parts.igem.org/Part:BBa_K4347010