Part:BBa_K1694023

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

Introduction:

Fig.1 Pcons+B0034+Lpp-OmpA-N+scFv(anti-VEGF)

By ligating the constitutive promoter (BBa_J23101), strong ribosome binding site (BBa_B0034) and Lpp-OmpA

(BBa_K1694002) connected to anti-VEGF (BBa_K1694003), we were able to display scFv(anti-VEGF) outside the E. coli cell membrane.

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.

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

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

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

2. Co-transform (Two plasmids)<bt> (1) Parts

Fig.4 Pcons+RBS+Lpp-OmpA-N+anti-VEGF

Fig.5 Pcons+RBS+RFP+Ter

Fig.6 Pcons+RBS+GFP+Ter

(2) Cell staining

After cloning the part of anti-VEGF, we were able to co-transform anti-VEGF 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-VEGF and expressed fluorescence protein.
To prove this, we conducted the cell staining experiment by using the co-transformed E. coli to detect VEGF in the cancer cell line.

Fig.7 As shown in the results, no red fluorescent E. coli bound on the cell’s surface as there were no specific scFv displayed around the E. coli.

Fig.8 There were red fluorescent anti-VEGF E. coli bound on the cell’s surfaces as the anti-VEGF probes on E. coli successfully detected and bound with VEGF.

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

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

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.11 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 13 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 9 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 13 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
  
• 1000
  COMPATIBLE WITH RFC[1000]