Part:BBa_K1694024

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

Introduction:

Fig.1 Pcons+B0034+Lpp-OmpA-N+scFv(Anti-EGFR)

By ligating the constitutive promoter (BBa_J23101), strong ribosome binding site (BBa_B0034) and Lpp-OmpA (BBa_K1694002) connected to Anti-EGFR (BBa_K1694004) , we were able to display the Anti-EGFR on the E. coli outer membrane continuously.

This year we want to create a customized platform, so we generated two libraries, Pcon+RBS+OmpA-scFv and Pcons+RBS+Fluorescence+Ter. From these libraries, we can select one plasmid per library to cotransform into the E. coli. Therefore, our customers can choose any scfv and any fluorescence protein. Our team will then co-transform the two plasmids, which help us tailor our product to satisfy the wishes of our customers.

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

Experiment:

1.Cloning

Fig.2The 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 assemble 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 part. The DNA sequence length of these parts is around 1100~1300 bp. In this PCR experiment, the scFv products size should be near at 1300~1500 bp. The Fig. 2 showed the correct size of the scFv, and proved that we successful ligated the scFv sequence onto an ideal backbone.

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

2.Co-transform (Two plasmids)

(1) Parts:

Fig.4 Pcons+RBS+Lpp-OmpA-N+Anti-EGFR

Fig.5 Pcons+RBS+RFP+Ter

Fig.6 Pcons+RBS+GFP+Ter

(2) Cell staining experiment:

After cloning the part of anti-EGFR, we were able to cotransform anti-EGFR with different fluorescence protein into our E. coli.
The next step was to prove that our co-transformed product has successfully displayed scFv of anti-EGFR and expressed fluorescence protein.
To verify this, we conducted the cell staining experiment by using cotransformed E. coli to detect the EGFR on the cancer cell line.

(3) Staining results：

Fig.7 As shown in the results, there were no red fluorescent E. colis bound on the cell’s surface because of the lack of specific-binding scFv displayed around the E.coli.

Fig.8 There were green fluorescent anti-EGFR E. colis bound on the cell’s surfaces, which indicated that the anti-EGFR on the surface of E. colis could successfully detect and bind on the EGFR.

Fig.9 As shown in the results, there were no green fluorescent E. colis binding on the cell’s surface because there is no specific scFv displayed around the E.coli.

Fig.10 There were red fluorescent anti-EGFR E. colis bound on the cell’s surfaces, which indicated that the anti-EGFR displayed by the E. coli could successfully detect and bind on the EGFR.

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.

Co-transform (Two plasmids)

Fig.11 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 18 hours. Thus, we can know that the E. Cotector can have maximum efficiency at this point.

Fig.12 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 16 hours. Thus, we can know that the E. Cotector can have maximum efficiency at this point.

Fig.13 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 6 hours. Thus, we can know that the E. Cotector can have maximum efficiency at this point.

Usage and Biology

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
  
• 1000
  COMPATIBLE WITH RFC[1000]