Latest revision as of 10:46, 22 September 2015

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 fluorescent proteins combination they want. We will co-transform the two plasmids, which helps us tailor our product to the wishes of our customers.

Introduction of basic parts:
Lpp-OmpA-N
Anti-HER2

Experiment

1.Cloning

Fig.2 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.

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.2 showed the correct size of this part, and proved that we successfully ligated the sequence onto an ideal backbone.

Fig.3 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 fluorescent 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 fluorescent 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.4 As shown in the results, no red fluorescent E. coli sticking on the cell’s surface as there were no specific scFv displayed around the E.coli.

Fig.5 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.6 As shown in the results, no green fluorescent E. coli bound on the cell’s surface as were no specific scFv displayed around the E.coli.

Fig.7 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 expression time by using the genetic algorithm (GA) in Matlab.
We want to characterize the actual kinetics of this Hill-function based model that accurately reflects protein expression time.
By using the differential function which was derived from these optimum parameters which were calculated by GA can help us to simulate the optimum protein expression. When we have the simulated protein expression rate, the graph of protein expression versus time can be drawn.Thus, we can find the optimum protein expression time. However, the simulated protein expression curve is slower than the experimental curve by one hour. Therefore, to find the most exact optimum protein expression time, we infer that subtracting one hour of the optimum protein expression time would be correct.

Co-transform

Fig.8 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.

Fig.9 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.

Fig.10 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