Composite

Part:BBa_K1694023

Designed by: CHIH-HSUAN HSU   Group: iGEM15_NCTU_Formosa   (2015-09-15)
Revision as of 07:45, 22 September 2015 by RayShing (Talk | contribs)

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.3The 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. 3 showed the correct size of the scFv, and proved that we successfully ligated the scFv sequence onto an ideal backbone.


Fig.4 Pcons+B0034+Lpp-OmpA-N+scFv(anti-VEGF)

1. Co-transform (Two plasmids)


(1) Parts:

Fig.7 Co-transform (Two plasmids)
Fig.8 Pcons+RBS+Lpp-OmpA-N+anti-VEGF
Fig.9 Pcons+RBS+RFP+Ter
Fig.10 Pcons+RBS+GFP+Ter


(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：

Fig.11 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.12 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.13 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.14 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.



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:

Fig.15 Transformation of single plasmid
Fig.16 Pcons+RBS+Lpp-OmpA-N+anti-VEGF+RBS+GFP+Ter


Fig.17 Pcons+RBS+Lpp-OmpA-N+anti-VEGF+RBS+amilCP+Ter


(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：



Fig.18 As results, no green fluorescent E. coli bound on the cell’s surface as there were no specific scFv displayed around the E. coli.
Fig.19 There are green fluorescent anti-VEGF E. coli stick on the cell’s surface as the anti-VEGF probes on E. coli successfully detect and bind with VEGF.
Fig.20 As results, no blue chromoprotein E. coli bound on the cell’s surface as there were no specific scFv displayed around the E. coli.
Fig.21 There were blue chromoprotein anti-VEGF E. coli bound on the cell’s surface as the anti-VEGF probes on E. coli successfully detected and bound with VEGF.

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


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


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Unknown
  • 12
    INCOMPATIBLE WITH RFC[12]
    Unknown
  • 21
    INCOMPATIBLE WITH RFC[21]
    Unknown
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 451
  • 1000
    COMPATIBLE WITH RFC[1000]


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