Part:BBa_K1694024
Pcons+B0034+Lpp-OmpA-N+scFv(Anti-EGFR)
Introduction:
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:
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.
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
- 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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