Part:BBa_K1694025

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

Introduction:

Fig.1 Pcons+RBS+Lpp-OmpA-N+anti-HER2

By ligating the constitutive promoter (BBa_J23101), strong ribosome binding site (BBa_B0034) and Lpp-OmpA (BBa_K1694002) connected to Anti-HER2 (BBa_K1694005) , we were able to display the Anti-EGFR on the E. coli outer membrane continuously.

This year we want to supply a customized platform. We provide two plasmids libraries of Pcon+RBS+OmpA-scFv and Pcons+RBS+Fluorescence+Ter for customers. Therefore,customers can choose any scFv and fluorescence proteins combination they want. We will co-transform the two plasmids, which helps us tailor our product to the wishes of our customers.

Experiment

1.Cloning
After assembling the DNA sequences from the basic parts, we recombined each Pcons+RBS+Lpp-OmpA-N+ScFv gene to pSB1C3 backbones and conducted a PCR experiment to check the size of each parts. The DNA sequence length of these parts is around 1100~1300 bp. In this PCR experiment, the PCR products' size should be near at 1300~1500 bp. The Fig. 3 showed the correct size of this part, and proved that we successfully ligated the sequence onto an ideal backbone.

Fig.3 The PCR result of the Pcons+B0034+Lpp-OmpA-N+ScFv. The DNA sequence length is around 1100~1300 bp, so the PCR products should appear at 1300~1500 bp.

Fig.4 Pcons+RBS+Lpp-OmpA-N+ScFv(anti-HER2)

2.Cell staining experiment
After cloning the part of anti-HER2, we were able to co-transform anti-HER2 with different fluorescence protein into our E. coli.
The next step was to prove that our co-transformed product have successfully displayed ScFv of anti-HER2 and expressed fluorescence protein.
To prove this, we conducted the cell staining experiment by using the co-transformed E. coli to detect the HER2 in the cancer cell line.

Fig.9 As results, no red fluorescent E. coli sticking on the cell’s surface as there were no specific scFv displayed around the E.coli.

Fig.10 There were red fluorescent anti-HER2 E. coli bound on the cell’s surfaces as the anti-HER2 probes on E. coli successfully detected and bound with HER2.

Fig.11 As results, no green fluorescent E. coli bound on the cell’s surface as were no specific scFv displayed around the E.coli.

Fig.12 There were green fluorescent anti-HER2 E. coli bound on the cell’s surfaces as the anti-HER2 probes on E. coli successfully detected and bound with HER2.

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 (Fig.1) (Fig.2) (Fig.3). Thus, we'll know the time of optimum production and the simulated one are fitted or not.

Co-transform

From this graph, the orange curve is the simulated protein expression. The blue curve is our experimental data. By comparing the orange curve to the blue curve, the blue one quite fits the simulation. The orange curve reaches peak after growing about 18 hours. Thus, we can know that the E.Cotector can have maximum efficiency at this point.

From this graph, the orange curve is the simulated protein expression. The blue curve is our experimental data. By comparing the orange curve to the blue curve, the blue one quite fits the simulation. The orange curve reaches peak after growing about 15 hours. Thus, we can know that the E.Cotector can have maximum efficiency at this point.

From this graph, the orange curve is the simulated protein expression. The blue curve is our experimental data. By comparing the orange curve to the blue curve, the blue one quite fits the simulation. The orange curve reaches peak after growing about 15 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
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 741
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

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