Part:BBa_K1694044

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

Introduction:

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

Transformation of single plasmid

To prove that our scFv can actually bind on to the antigens on carcinoma cells, we connected each scFv with a different fluorescent protein. Therefore, we can use fluorescent microscopes to clearly observe, if the E. coli has produced scFv proteins. Currently, we have built three different scFv connected with their respective fluorescent proteins, which are anti-VEGF+GFP, anti-EGFR+RFP, anti-HER2+BFP. The process of cell staining 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.

Introduction to basic parts:
Lpp-OmpA-N
Anti-EGFR

Experiment

1.Cloning

Fig.2 The 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 we assemble the DNA sequences from the basic parts, we recombined each Pcons+B0034+Lpp-OmpA-N+scFv(anti-EGFR)+B0030+RFP+J61048 gene to pSB1C3 backbones and conducted PCR experiments to ensure the size of each of the parts. The DNA sequence length of the these parts are around 2000~2200 bp. For PCR experiments, the size of products should be approximately 2200~2400 bp. The Fig.2 below shows the correct size of this part, and proved that we successful ligated the sequence onto an ideal backbone.

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

2. Transformation of single plasmid

Fig.4 Transformation of single plasmid

(1) Cell staining experiment: After inserting a part of the scFv gene into our E. coli, we were going to prove that our detectors have successfully displayed a functional 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 on the SKOV-3 cancer cell lines. SKOV-3 is a kind of epithelial cell that expressed markers such as EGFR.

(2) Staining results：

Fig.5 As a result, 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.6 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.

Modeling

In the modeling part, we discovered the optimum protein production time by using the genetic algorithm in Matlab.
We want to characterize the actual kinetics of this Hill-function based model that accurately reflects protein production time.
When we have the simulated protein production rate, the graph of protein production versus time can be drawn. Thus, we get the optimum protein production time Compared with the simulated protein production rate of time, our experiment data quite fit the simulation.

Fig.7 From this graph, the orange curve is the simulated protein expression. The blue curve is our experimental data. By comparing the orange curve and the blue curve, the blue curve has a close fit to the orange in the simulation. The orange curve reaches peak after growing about 17 hours. Thus, we can know that the E.Cotector can have maximum efficiency at this point.

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 1987
  Illegal AgeI site found at 1828
  Illegal AgeI site found at 1940
  
• 1000
  COMPATIBLE WITH RFC[1000]