Revision as of 19:52, 19 September 2015

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

Introduction:

Fig.1 Pcons+B0034+Lpp-OmpA-N+scFv(anti-EGFR)+B0030+GFP+J61048

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. Moreover, we built combinations of each scFv connected with GFP.

Experiment

Fig.2The PCR result of the Pcons+B0034+Lpp-OmpA-N+scFv(anti-EGFR)+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-EGFR)+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.2 showed the correct size of this part, and proved that we successful ligated the sequence onto an ideal backbone.

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.

Fig.3 Pcons+B0034+Lpp-OmpA-N+scFv(anti-EGFR)+B0030+GFP+J61048

Sequence and Features