Part:BBa_K4387982:Design

Bivalent nanobody expression with the HlyA secretion signal (biv. VHH#3E)

Assembly Compatibility:

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

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2091
  Illegal BglII site found at 2670
  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1660
  Illegal AgeI site found at 979
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

The inducible pBAD system was chosen to better characterize nanobody production under controlled conditions. This composite part required for the inducible anti-TNFα nanobody expression was ordered from Twist Bioscience, integrated in a high copy number backbone (colE1). The end goal is the exchange of the pBAD system by our NO-sensing device (BBa_K4387978). The Hemolysin A secretion machinery (BBa_K4387987) was ordered from Twist Bioscience in a medium copy number backbone (p15A) and allowed the secretion of the nanobodies via the HlyA one-step secretion system.

Source

The amino acid sequences of the individual nanobodies were directly taken from the patent mentioned in the description. [1] Sequences were then converted to DNA and codon optimized for E. coli strains. The sequences for the secretion system were obtained from a paper by Ruano-Gallego et al. [2].

References

• [1] Silence, Karen, Lauwereys, Marc, De Haard, Hans, et al. "Single domain antibodies directed against tumour necrosis factor-alpha and uses therefor", Int. Publication Number: WO 2004/041862 A2, 21 May 2004
• [2] Ruano-Gallego, D., Fraile, S., Gutierrez, C. et al. Screening and purification of nanobodies from E. coli culture supernatants using the hemolysin secretion system. Microb Cell Fact 18, 47 (2019). https://doi.org/10.1186/s12934-019-1094-0