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Part:BBa_K1694023
Pcons+B0034+Lpp-OmpA-N+scFv(Anti-VEGF)
Introduction:
By ligating the constitutive promoter (BBa_J23101), strong ribosome binding site (BBa_B0034) and Lpp-OmpA-scFv, we were able to display scFv(Anti-VEGF) outside the E.coli cell membrane.
This year we want to provide a customized platform. We provide two libraries of Pcon+RBS+OmpA-scFv and Pcons+RBS+Fluorescence+Ter into E. coli. Therefore, our customers can choose any scfv and any fluorescence protein. Our team will then co-transform the two plasmids, which helps us tailor our product to the wishes of our customers.
Experiment
1.Cloning
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 of the parts. 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. 3 showed the correct size of the scFv, and proved that we successful ligated the scFv sequence onto an ideal backbone.
1. Co-transform (Two plasmids)
This year we want to provide a customized platform. We provide two libraries of Pcon+RBS+OmpA-scFv and Pcons+RBS+Fluorescence+Ter into E. coli. Therefore, our customers can choose any scfv and any fluorescence protein. Our team will then co-transform the two plasmids, which helps us tailor our product to the wishes of our customers.
(1) Parts:
(2) Cell staining experiment:
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.
(3) Staining results:
2. Transformation of single plasmid
To prove that our scFv can actually bind on to the antigen on cancer cells, we connected each scFv with a different fluorescence protein. Therefore, we could use fluorescence microscope to clearly observe if the E. coli has produced scFv proteins. Currently, we built three different scFv connected with their respectively fluorescence protein. 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.
(1) Parts:
(2) 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-VEGF. To prove this, we have decided to undergo the cell staining experiment by using our E. coli to detect the VEGF in the SKOV-3 cancer cell lines. SKOV-3 is a kind of epithelial cell that expressed markers such as VEGF.
(3) Staining results:
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
![](/wiki/images/d/db/Anti-VEGF-GFP.jpg)
![](/wiki/images/8/86/VEGF-RFP.png)
![](/wiki/images/0/0b/VEGF-BFP.png)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 451
- 1000COMPATIBLE WITH RFC[1000]
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