Part:BBa_K3059621:Design

csgEFG, lacks an RBS before csgE

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 382
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 382
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 382
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 382
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 382
  Illegal AgeI site found at 679
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

This part was isolated from the E. coli MG1655 genome via PCR. Annealing sequences for primers were sourced from "A Synthetic Circuit for Mercury Bioremediation Using Self-Assembling Functional Amyloids" (Tay, Nguyen, Joshi 2017). These sequences were verified as genomic using NCBI Blast. Primer overhangs were designed to add sticky ends and BsaI cutsites to this basic part to create a composite part, 3G Assembly-compatible csgBAC (+22 bp upstream region). Primer overhangs also added 40-basepair "Pad" adapter sequences, which allow for insertion into William & Mary's Pad 1C3 cloning backbone via Gibson assembly.

Source

This part required primer design for genomic PCR. Annealing sequences were sourced from "A Synthetic Circuit for Mercury Bioremediation Using Self-Assembling Functional Amyloids" (Tay, Nguyen, Joshi 2017). These sequences were verified as genomic using NCBI Blast. Primer overhangs were designed to add sticky ends, BsaI cutsites, and Pad adapter sequences (for Gibson into Pad 1C3, a cloning backbone) to this basic part to create a composite part, 3G Assembly-compatible csgEFG.

References

1. Barnhart, M. M., & Chapman, M. R. (2006). Curli biogenesis and function. Annual review of microbiology, 60, 131–147. doi:10.1146/annurev.micro.60.080805.142106

2. Tay, P. K. R., Nguyen, P. Q., & Joshi, N. S. (2017). A Synthetic Circuit for Mercury Bioremediation Using Self-Assembling Functional Amyloids. ACS Synthetic Biology, 6(10), 1841–1850. doi: 10.1021/acssynbio.7b00137