Part:BBa_K1694034

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 fluorescent protein. Therefore we could use fluorescent microscope to clearly observe if the E. coli has produced scFv proteins. Currently,we built three different scFv connected with their respectively fluorescent 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.

Introduction to basic parts:

For more information on the modification of Lpp-OmpA-N, please click here.

Anti-EGFR

Experiment

1.Cloning

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.

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

2. Transformation of single plasmid

Fig.4 Transformation of single plasmid

(1) 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.

(2) Staining results：

Fig.5 As shown in the results, there were no green fluorescent E. coli bound on the cell’s surface as there were no specific scFv displayed around the E. coli.

Fig.6 There were green fluorescent anti-EGFR E. coli bound on the cell’s surface as the anti-EGFR probes on E. coli successfully detected and bound with EGFR.

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Modeling

In the modeling part, we discover 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 quite fit 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 2001
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1917