Part:BBa_K1694033

Pcons+B0034+Lpp-OmpA-N+scFv(Anti-VEGF)+B0030+GFP+J61048

Introduction:

Fig.1Pcons+RBS+Lpp-OmpA-N+Anti-VEGF+RBS+GFP+Ter

Transformation of single plasmid

To prove that our scFv can actually bind on to the antigen on cancer cells, we connected each scFv with a different fluorescence protein. Therefore we could use fluorescence microscope to clearly observe if the E. coli has produced scFv proteins. Currently,we built three different scFv connected with their respectively fluorescence protein, which are Anti-VEGF+GFP, Anti-EGFR+RFP, Anti-HER2+BFP. When applied on cell staining, we can identify the antigen distribution on cancer cells by observing the fluorescence. Furthermore, if we use the three scFv simultaneously, we can also detect multiple markers.

Experiment

Fig.3The PCR result of the Pcons+B0034+Lpp-OmpA-N+scFv(Anti-VEGF)+B0030+GFP+J61048. The DNA sequence length is around 2000~2200 bp, so the PCR products should appear at 2200~2400 bp.

After assemble the DNA sequences from the basic parts, we recombined each Pcons+B0034+Lpp-OmpA-N+scFv(Anti-VEGF)+B0030+GFP+J61048 gene to PSB1C3 backbones and conducted a PCR experiment to check the size of each of the parts. The DNA sequence length of the these parts are around 2000~2200 bp. In this PCR experiment, the PCR products size should be near at 2200~2400 bp. The Fig.3 showed the correct size of this part, and proved that we successful ligated the sequence onto an ideal backbone.

Fig.4Pcons+B0034+Lpp-OmpA-N+scFv(anti-VEGF)+B0030+GFP+J61048

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 451
  Illegal NgoMIV site found at 2013
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1929