Part:BBa_K1694035
Pcons+B0034+Lpp-OmpA-N+scFv(Anti-HER2)+B0030+GFP+J61048
Introduction:
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. We also built combinations each scFv connected with green fluorescence protein.
Experiment
1.Cloning
After assemble the DNA sequences from the basic parts, we recombined each Pcons+B0034+Lpp-OmpA-N+scFv(Anti-HER2)+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 1900~2100 bp. In this PCR experiment, the PCR products size should be near at 2100~2300 bp. The Fig.3 showed the correct size of this part, and proved that we successful ligated the sequence onto an ideal backbone.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 741
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 451
Illegal NgoMIV site found at 1846 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1762
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