Composite

Part:BBa_K1694024

Designed by: CHIH-HSUAN HSU   Group: iGEM15_NCTU_Formosa   (2015-09-15)
Revision as of 02:17, 19 September 2015 by SherryNCTU (Talk | contribs)

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

Introduction:

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By ligating the constitutive promoter (BBa_J23101), strong ribosome binding site (BBa_B0034) and Lpp-OmpA-scFv, we were able to display scFv on the E. coli outer membrane continuously.
Having this part, we can co-transform with other parts in order to produce color as the detection signal.

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 receiving the DNA sequences from the gene synthesis company, we recombined each scFv gene to PSB1C3 backbones and conducted a PCR experiment to check the size of each of the scFvs. The DNA sequence length of the scFvs are around 600~800 bp. In this PCR experiment, the scFv products size should be near at 850~1050 bp. The Fig. showed the correct size of the scFv, and proved that we successful ligated the scFv sequence onto an ideal backbone.

2.Co-transform

Fig.6 Pcons+RBS+Lpp-OmpA-N+Anti-EGFR
Fig.7 Pcons+RBS+RFP+Ter
Fig.8 Pcons+RBS+GFP+Ter

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


Fig.9 ~ Fig. 12 are our staining results：
Negative control:
There are red and green fluorescent anti-EGFR E. coli stick on the cell’s surfaces as the anti-EGFR probes on E. colis successfully detect and bind with EGFR.

Fig.9 As results,there is no green fluorescent E. coli stick on the cell’s surface as there is no specific scFv displayed around the E.coli.
Fig.10 As results,there is no red fluorescent E. coli stick on the cell’s surface as there is no specific scFv displayed around the E.coli.


Fig.11 There are green fluorescent anti-EGFR E. colis stick on the cell’s surfaces as the anti-EGFR probes on E. colis successfully detect and bind with EGFR.
Fig.12 There are red fluorescent anti-EGFR E. colis stick on the cell’s surfaces as the anti-EGFR probes on E. colis successfully detect and bind with EGFR.

cell staining experiment:


After creating the part of anti-EGFR，we are able to co-transform them with different fluorescent parts into our E. Cotector.
The next step is to prove that our co-transformed product have successfully displayed scFv of anti-EGFR and expressed fluorescent part.
To prove this, we have decided to undergo the cell staining experiment by using our E. Cotector to detect the EGFR in the cell lines.
Each type of E. Cotector has been co-transformed with two different fluorescent colors ---RFP and GFP

Procedure：
First of all, the main materials that we needed are red and green fluorescent of co-transform E.Coli with scFv of anti-EGFR, red green blue fluorescent E. coli without scFv and the cancer cell line – SKOV-3 that expressed EGFR, for staining used.
SKOV-3 is a kind of epithelial cell that expressed markers such as EGFR.
After injecting E.Coli into the wells, we had to shake the plate in darkness for 45minutes. After staining for 45 minutes, we will wash away the unbind E.Coli with PBS solution for a few times before observing the staining result under fluorescent microscope.


Below are our staining result：
Negative control:
As results,there is no green fluorescent E. coli stick on the cell’s surface as there is no specific scFv displayed around the E. coli .
There are red and green fluorescent anti-EGFR E.Cotectors stick on the cell’s surfaces as the anti-EGFR probes on E.Cotectors successfully detect and bind with 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 (Fig.1) (Fig.2) (Fig.3). Thus, we get the optimum protein production time Compared with the simulated protein production rate of time, our experiment data quite fit the simulation.

From this graph, the protein expression reaches peak after growing about 15 hours. This means that the E. Cotector can have maximum efficiency at this point
From this graph, the protein expression reaches peak after growing about 18 hours. This means that the E. Cotector can have maximum efficiency at this point
From this graph, the protein expression reaches peak after growing about 15 hours. This means 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]


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