Part:BBa_K1607011:Design

The SCFV of anti-p185her2/neu antibody chA21 linked with EGFP by a (Gly4Ser)3 linker

Assembly Compatibility:

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

  Illegal BglII site found at 370
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Source

The scFv CDS was synthesized by LCR assmebly using 36 oilgos

see oligos and protocols

The eGFP CDS was obtained using PCR. Primers:

ggaggcggaggcagcggtggcggtggaagcATGGTGAGCAAGGGCga (eGFP-F)

GGTCTAGAGGCTTGTACAGCTCGTCCAT (eGFP-R)

The eGFP CDS and the scFv CDS were connected by SOE PCR

Primers:

step1:

CGGAATTCatgGGTTCTACTCAAG (SCFV-F)

gctgcctccgcctccagaaccgccaccgccAGATTGACAATCTTCagaaga (SCFV-R)

ggaggcggaggcagcggtggcggtggaagcATGGTGAGCAAGGGCga (eGFP-F)

GGTCTAGAGGCTTGTACAGCTCGTCCAT (eGFP-R)

step2:

SCFV-F and eGFP-R

Standard Biobrick prefix and suffix were then added to the sequence by PCR, replacing the EcoRI site and XbaI site which were originally designed for yeast expression.

Primers:

GAATTCGCGGCCGCTTCTAGatgGGTTCTACTCAAGttt (F)

TACTAGTAGCGGCCGCTGCAGCTTGTACAGCTCGTCCAT(R)

Design Notes

This part is designed for FRET analysis. An Mcherry will be fused with the antigen, and by measuring the FRET efficiency we can detect the antibody-antigen interaction.

References

[1] Zhou H, Zha Z, Liu Y, et al. Structural Insights into the Down-regulation of Overexpressed p185her2/neu Protein of Transformed Cells by the Antibody chA21[J]. Journal of Biological Chemistry, 2011, 286(36): 31676-31683.