Part:BBa_K1694054

Pcons+B0034+Lpp-OmpA-N+scFv(Anti-EGFR)+B0034+amilCP+B0015

2. 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. 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.

(1) Parts:

Fig.13 Transformation of single plasmid

Fig.14 Pcons+RBS+Lpp-OmpA-N+Anti-EGFR+RBS+RFP+Ter

Fig.15 Pcons+RBS+Lpp-OmpA-N+Anti-EGFR+RBS+GFP+Ter

(2) Cell staining experiment: After creating the part of scFv and transforming them into our E. coli, we were going to prove that our detectors have successfully displayed scFv of anti-EGFR. To prove this, we have decided to undergo the cell staining experiment by using our E. coli to detect the EGFR in the SKOV-3 cancer cell lines. SKOV-3 is a kind of epithelial cell that expressed markers such as EGFR.

(3) Staining results：

Fig.16 As results,there is no red fluorescent E. coli stick on the cell’s surface as there is no specific scFv displayed around the E. coli.

Fig.17 There are green fluorescent anti-EGFR E. coli stick on the cell’s surface as the anti-EGFR probes on E. coli successfully detect and bind with EGFR.

Fig.18 As results,there is no green fluorescent E. coli stick on the cell’s surface as there is no specific scFv displayed around the E. coli.

Fig.19 There are green fluorescent anti-EGFR E. coli stick on the cell’s surface as the anti-EGFR probes on E. coli successfully detect and bind with EGFR.

Fig.20 As results,there is no green fluorescent E. coli stick on the cell’s surface as there is no specific scFv displayed around the E. coli.

Fig.21 There are blue chromoprotein anti-EGFR E. coli stick on the cell’s surface as the anti-EGFR probes on E. coli successfully detect and bind with EGFR.

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
  
• 1000
  COMPATIBLE WITH RFC[1000]