Difference between revisions of "Part:BBa K1694024"
 
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<h1>'''Introduction:'''</h1>
 
<h1>'''Introduction:'''</h1>
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[[File:EGFRHALF.png|600px|thumb|center|Pcons+B0034+Lpp-OmpA-N+scFv(Anti-EGFR) ]]  
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[[File:EGFRHALF.png|600px|thumb|center|'''Fig.1''' Pcons+B0034+Lpp-OmpA-N+scFv(Anti-EGFR) ]]  
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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.
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By ligating the constitutive promoter (BBa_J23101), strong ribosome binding site (BBa_B0034) and Lpp-OmpA  
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<br>
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<html><a href="https://parts.igem.org/Part:BBa_K1694002">(BBa_K1694002)</a>  
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Having this part, we can co-transform with other parts in order to produce color as the detection signal.
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</html>
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<br><br>
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connected to Anti-EGFR
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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.
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<html><a href="https://parts.igem.org/Part:BBa_K1694004">(BBa_K1694004)</a></html> , we were able to display the Anti-EGFR on the E. coli outer membrane continuously.  
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<h1>'''Experiment:'''</h1>
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<p style="font-size:120%">'''1.Cloning'''</p>
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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.
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<p style="font-size:120%">'''2.Co-transform'''</p>
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[[File:EGFRHALF.png|600px|thumb|center|'''Fig.6''' Pcons+RBS+Lpp-OmpA-N+Anti-EGFR]]
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[[File:Pcons+RBS+RFP+Ter.png|600px|thumb|center|'''Fig.7''' Pcons+RBS+RFP+Ter]]
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[[File:GFP2015.png|600px|thumb|center|'''Fig.8''' Pcons+RBS+GFP+Ter]]
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'''Cell staining experiment:'''
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<br>
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After cloning the part of anti-EGFR, we were able to co-transform anti-EGFR with different fluorescence protein into our ''E. coli''. <br>
+
−
The next step was to prove that our co-transformed product have successfully displayed scFv of anti-EGFR and expressed fluorescence protein.
+
−
<br>
+
−
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.  
+
 
<br>
 
<br>
 
<br>
 
<br>
 +
This year we want to create a customized platform, so we generated two libraries, Pcon+RBS+OmpA-scFv and Pcons+RBS+Fluorescence+Ter. From these libraries, we can select one plasmid per library to cotransform into the E. coli. Therefore, our customers can choose any scfv and any fluorescent protein. Our team will then co-transform the two plasmids, which help us tailor our product to satisfy the wishes of our customers.
  
 +
<html>
 +
Introduction of basic parts:
 
<br>
 
<br>
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<b>Fig.9</b> ~<b> Fig. 12 </b>are our staining results：
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<a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1694002"> Lpp-OmpA-N</a><br>
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<br>
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<a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1694004"> Anti-EGFR </a><br>
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Negative control:
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</html>
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<br>
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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.
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[[File:NGFP.png|400px|thumb|left|'''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''. ]]
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[[File:NRFP.png|400px|thumb|left|'''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''. ]]
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−
<br>
 
  
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<div style="display: block; height: 520pt;">
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<h1>'''Experiment:'''</h1>
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[[File:EGFPGFPCELLCO.png|400px|thumb|left|'''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.]]
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'''1.Cloning'''
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[[File:EGFPRFPCELLCO.png|400px|thumb|left|'''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.]]
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[[File:PROCPCR.png|200px|thumb|left|'''Fig.2''' The 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 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 part. 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.2''' showed the correct size of the scFv, and proved that we successful ligated the scFv sequence onto an ideal backbone.
 +
 
 +
<div style="display: block; height: 400pt;">
 +
[[File:PROCplate.png|600px|thumb|center|'''Fig.3''' Pcons+B0034+Lpp-OmpA-N+scFv(anti-EGFR)]]  
 
</div>
 
</div>
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<p style="font-size:120%">'''cell staining experiment:'''</p>
+
 
 +
'''2.Cell staining'''
 +
 
 +
After cloning the part of anti-EGFR, we were able to cotransform anti-EGFR with different fluorescent protein into our ''E. coli''. <br>
 +
The next step was to prove that our co-transformed product has successfully displayed scFv of anti-EGFR and expressed fluorescent protein.
 
<br>
 
<br>
−
After creating the part of anti-EGFR，we are able to co-transform them with different fluorescent parts into our E. Cotector. <br>
+
To verify this, we conducted the cell staining experiment by using cotransformed ''E. coli'' to detect the EGFR on the cancer cell line.  
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The next step is to prove that our co-transformed product have successfully displayed scFv of anti-EGFR and expressed fluorescent part.  
+
 
<br>
 
<br>
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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.
 
 
<br>
 
<br>
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Each type of E. Cotector has been co-transformed with two different fluorescent colors ---RFP and GFP
 
 
<br>
 
<br>
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<br>
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[[File:Co3.png|400px|thumb|left|'''Fig.4''' As shown in the results, there were no red fluorescent ''E. colis'' bound on the cell’s surface because of the lack of specific-binding scFv displayed around the ''E.coli''. ]]
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'''Procedure：'''
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[[File:EGFPGFPCELLCO.png|400px|thumb|left|'''Fig.5''' There were green fluorescent anti-EGFR ''E. colis'' bound on the cell’s surfaces, which indicated that the anti-EGFR on the surface of ''E. colis'' could successfully detect and bind on the EGFR.]]
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<br>
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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.
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<br>
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SKOV-3 is a kind of epithelial cell that expressed markers such as EGFR.
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<br>
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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.
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<br>
 
<br>
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Below are our staining result：
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<div style="display: block; height: 520pt;">
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<br>
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[[File:NGFP.png|400px|thumb|left|'''Fig.6''' As shown in the results, there were no green fluorescent ''E. colis'' binding on the cell’s surface because there is no specific scFv displayed around the ''E.coli''. ]]
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Negative control:
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[[File:EGFPRFPCELLCO.png|400px|thumb|left|'''Fig.7''' There were red fluorescent anti-EGFR ''E. colis'' bound on the cell’s surfaces, which indicated that the anti-EGFR displayed by the ''E. coli'' could successfully detect and bind on the EGFR.]]
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<br>
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</div>
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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 .  
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<br>
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<h1>'''Modeling'''</h1>
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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.
+
  
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<p style="font-size:120%">'''Modeling:'''</p>
 
 
In the modeling part, we discover optimum protein production time by using the genetic algorithm in Matlab.
 
In the modeling part, we discover optimum protein production time by using the genetic algorithm in Matlab.
 
<br>
 
<br>
 
We want to characterize the actual kinetics of this Hill-function based model that accurately reflects protein production time.
 
We want to characterize the actual kinetics of this Hill-function based model that accurately reflects protein production time.
 
<br>
 
<br>
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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
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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.
 
Compared with the simulated protein production rate of time, our experiment data quite fit the simulation.
 +
 
<br>
 
<br>
 +
<p style="font-size:120%">'''Co-transform (Two plasmids)'''</p>
 +
 
<br>
 
<br>
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From this graph, the protein expression reaches peak after growing about 15 hours.
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This means that the E. Cotector can have maximum efficiency at this point
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[[File:Anti-EGFR-RFP.jpg|900px|thumb|center|'''Fig.8'''  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 18 hours.
 +
Thus, we can know that the E. Cotector can have maximum efficiency at this point.]]
 +
 
 
<br>
 
<br>
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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
+
[[File:anti-EGFR-GFP.jpg|900px|thumb|center|'''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 and the blue curve, the blue curve quite fit the simulation.
 +
The orange curve reaches peak after growing about 16 hours.
 +
Thus, we can know that the E. Cotector can have maximum efficiency at this point.]]
 +
 
 
<br>
 
<br>
−
From this graph, the protein expression reaches peak after growing about 15 hours.
+
[[File:Anti-EGFR-BFP.jpg|900px|thumb|center|'''Fig.10'''  From this graph, the orange curve is the simulated protein expression. The blue curve is our experimental data.
−
This means that the E. Cotector can have maximum efficiency at this point
+
By comparing the orange curve and the blue curve, the blue curve quite fit the simulation.
 +
The orange curve reaches peak after growing about 6 hours.
 +
Thus, we can know that the E. Cotector can have maximum efficiency at this point.]]
 +
 
 
<br>
 
<br>
  
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
  

Latest revision as of 10:51, 22 September 2015

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

Introduction:

Fig.1 Pcons+B0034+Lpp-OmpA-N+scFv(Anti-EGFR)

By ligating the constitutive promoter (BBa_J23101), strong ribosome binding site (BBa_B0034) and Lpp-OmpA (BBa_K1694002) connected to Anti-EGFR (BBa_K1694004) , we were able to display the Anti-EGFR on the E. coli outer membrane continuously.

This year we want to create a customized platform, so we generated two libraries, Pcon+RBS+OmpA-scFv and Pcons+RBS+Fluorescence+Ter. From these libraries, we can select one plasmid per library to cotransform into the E. coli. Therefore, our customers can choose any scfv and any fluorescent protein. Our team will then co-transform the two plasmids, which help us tailor our product to satisfy the wishes of our customers.

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


Experiment:

1.Cloning

Fig.2 The 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 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 part. 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.2 showed the correct size of the scFv, and proved that we successful ligated the scFv sequence onto an ideal backbone.

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

2.Cell staining

After cloning the part of anti-EGFR, we were able to cotransform anti-EGFR with different fluorescent protein into our E. coli.
The next step was to prove that our co-transformed product has successfully displayed scFv of anti-EGFR and expressed fluorescent protein.
To verify this, we conducted the cell staining experiment by using cotransformed E. coli to detect the EGFR on the cancer cell line.


Fig.4 As shown in the results, there were no red fluorescent E. colis bound on the cell’s surface because of the lack of specific-binding scFv displayed around the E.coli.
Fig.5 There were green fluorescent anti-EGFR E. colis bound on the cell’s surfaces, which indicated that the anti-EGFR on the surface of E. colis could successfully detect and bind on the EGFR.


Fig.6 As shown in the results, there were no green fluorescent E. colis binding on the cell’s surface because there is no specific scFv displayed around the E.coli.
Fig.7 There were red fluorescent anti-EGFR E. colis bound on the cell’s surfaces, which indicated that the anti-EGFR displayed by the E. coli could successfully detect and bind on the 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. 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 (Two plasmids)


Fig.8 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 18 hours. Thus, we can know that the E. Cotector can have maximum efficiency at this point.


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 and the blue curve, the blue curve quite fit the simulation. The orange curve reaches peak after growing about 16 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 6 hours. Thus, we can know that the E. Cotector can have maximum efficiency at this point.


Usage and Biology

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]