Part:BBa_K1694045

Pcons+B0034+Lpp-OmpA-N+scFv(Anti-HER2)+B0030+BFP+J61048

Introduction:

Fig.1 Pcons+B0034+Lpp-OmpA-N+scFv(anti-HER2)+B0030+BFP+J61048

Transformation of single plasmid

To prove that our scFv can actually bind on to the antigen on carcinoma cells, we connected each scFv with a different fluorescent protein. Therefore we can use fluorescent microscope 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 of basic parts:
Lpp-OmpA-N
Anti-HER2
OmpA-Anti-HER2

Experiment

1.Cloning

Fig.2 The PCR result of the Pcons+B0034+Lpp-OmpA-N+scFv(anti-HER2)+B0030+BFP+J61048. The DNA sequence length is around 1900~2100 bp, so the PCR products should appear at 2100~2300 bp.

After assemble the DNA sequences from the basic parts, we recombined each Pcons+B0034+Lpp-OmpA-N+scFv(Anti-HER2)+B0030+BFP+J61048 gene to pSB1C3 backbones and conducted a PCR experiments to ensure the size of each of the parts. The DNA sequence length of the these parts are around 1900~2100 bp. For PCR experiments, the size of products should be approximately 2100~2300 bp. The Fig.2 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-HER2)+B0030+BFP+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-HER2. To prove this, we have decided to undergo the cell staining experiment by using our E. coli to detect the HER2 on the SKOV-3 cancer cell lines. SKOV-3 is a kind of epithelial cell that expressed markers such as HER2.

(2) Staining results：

Fig.5 As results,there is no blue fluorescent E. coli stick on the cell’s surface as there is no specific scFv displayed around the E. coli.

Fig.6 There are blue fluorescent anti-HER2 E. coli stick on the cell’s surface as the anti-HER2 probes on E. coli successfully detect and bind with HER2.

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, we speculated that experimental error cause the undesired result.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Unknown
• 12
  INCOMPATIBLE WITH RFC[12]

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• 21
  INCOMPATIBLE WITH RFC[21]

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• 23
  INCOMPATIBLE WITH RFC[23]

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• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 451
  Illegal NgoMIV site found at 1831
  
• 1000
  COMPATIBLE WITH RFC[1000]