Difference between revisions of "Part:BBa K1694023"

 
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<h1>'''Introduction:'''</h1>
 
<h1>'''Introduction:'''</h1>
[[File:PROVEGF.png|600px|thumb|center|'''Fig.1''' Pcons+B0034+Lpp-OmpA-N+scFv(Anti-VEGF)]]
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[[File:PROVEGF.png|600px|thumb|center|'''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  
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.
+
(<html><a href="https://parts.igem.org/Part:BBa_K1694002">BBa_K1694002</a>) connected to anti-VEGF (<a href="https://parts.igem.org/Part:BBa_K1694003">BBa_K1694003</a>)</html>, we were able to display scFv(anti-VEGF) outside the<em> E. coli</em> cell membrane.
 
<br><br>
 
<br><br>
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.
+
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.
  
<h1>'''Experiment'''</h1>
+
<html>
<p style="font-size:120%">'''1.Cloning'''</p>
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Introduction of basic parts:
[[File:PROBPCR.png|200px|thumb|left|'''Fig.3'''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.]]
+
<br>
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.
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<a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1694002"> Lpp-OmpA-N</a><br>
 +
<a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1694003"> Anti-VEGF </a><br>
  
 +
</html>
  
[[File:PROBplate.png|600px|thumb|center|'''Fig.4''' Pcons+B0034+Lpp-OmpA-N+scFv(Anti-VEGF)]]
 
  
<p style="font-size:120%">'''1. Co-transform (Two plasmids)'''</p>
 
  
 +
<h1>'''Experiment'''</h1>
 +
'''1.Cloning'''
  
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.
+
[[File:PROBPCR.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 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.2''' showed the correct size of the scFv, and proved that we successfully ligated the scFv sequence onto an ideal backbone.
  
<br>
+
<div style="display: block; height: 400pt;">
 
+
[[File:PROBplate.png|600px|thumb|center|'''Fig.3''' Pcons+B0034+Lpp-OmpA-N+scFv(anti-VEGF)]]
''' (1) Parts:'''
+
</div>
 
+
'''2. Co-transform (Two plasmids)'''<br>
[[File:COTR.png|600px|thumb|center|'''Fig.7''' Co-transform (Two plasmids)]]
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''' (1) Parts'''
[[File:PROB.png|600px|thumb|center|'''Fig.8''' Pcons+RBS+Lpp-OmpA-N+Anti-VEGF]]
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[[File:PROB.png|600px|thumb|center|'''Fig.4''' Pcons+RBS+Lpp-OmpA-N+anti-VEGF]]
[[File:Pcons+RBS+RFP+Ter.png|600px|thumb|center|'''Fig.9''' Pcons+RBS+RFP+Ter]]  
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[[File:Pcons+RBS+RFP+Ter.png|600px|thumb|center|'''Fig.5''' Pcons+RBS+RFP+Ter]]  
[[File:GFP2015.png|600px|thumb|center|'''Fig.10''' Pcons+RBS+GFP+Ter]]
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[[File:GFP2015.png|600px|thumb|center|'''Fig.6''' Pcons+RBS+GFP+Ter]]
  
 
<br>
 
<br>
''' (2) Cell staining experiment:'''
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''' (2) Cell staining '''
  
 
After cloning the part of anti-VEGF, we were able to co-transform anti-VEGF with different fluorescence protein into our ''E. coli''. <br>
 
After cloning the part of anti-VEGF, we were able to co-transform anti-VEGF with different fluorescence protein into our ''E. coli''. <br>
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<br>
 
<br>
 
<br>
 
<br>
''' (3) Staining results:'''
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[[File:Co3.png|400px|thumb|left|'''Fig.7''' 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''. ]]
<br>
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[[File:VEGFRFPCELLCO.png|400px|thumb|left|'''Fig.8''' 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.]]  
[[File:Co3.png|400px|thumb|left|'''Fig.11''' 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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[[File:VEGFRFPCELLCO.png|400px|thumb|left|'''Fig.12''' There are red fluorescent anti-VEGF ''E. coli'' stick on the cell’s surfaces as the anti-VEGF probes on ''E. coli'' successfully detect and bind with VEGF.]]  
+
  
 
<br>
 
<br>
 
<div style="display: block; height: 520pt;">
 
<div style="display: block; height: 520pt;">
[[File:NGFP.png|400px|thumb|left|'''Fig.13''' 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:NGFP.png|400px|thumb|left|'''Fig.9''' 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''. ]]
[[File:VEGFGFPCELLCO.png|400px|thumb|left|'''Fig.14''' There are green fluorescent anti-VEGF ''E. coli'' stick on the cell’s surfaces as the anti-VEGF probes on ''E. coli'' successfully detect and bind with VEGF.]]
+
[[File:VEGFGFPCELLCO.png|400px|thumb|left|'''Fig.10''' 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.]]
 
</div>
 
</div>
  
 
 
 
<p style="font-size:120%">'''2. Transformation of single plasmid'''</p>
 
 
 
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.
 
 
<br>
 
''' (1) Parts:'''
 
[[File:TR.png|600px|thumb|center|'''Fig.15''' Transformation of single plasmid]]
 
 
[[File:VEGFGFP.png|900px|thumb|center|'''Fig.16''' Pcons+RBS+Lpp-OmpA-N+Anti-VEGF+RBS+GFP+Ter]]
 
<br>
 
[[File:VEGFCP1.png|900px|thumb|center|'''Fig.17''' Pcons+RBS+Lpp-OmpA-N+Anti-VEGF+RBS+amilCP+Ter]]
 
<br>
 
 
''' (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.
 
<br>
 
<br>
 
''' (3) Staining results:'''
 
 
<br>
 
 
 
<div style="display: block; height: 250pt;">
 
 
[[File:Ng.png|400px|thumb|left|'''Fig.18''' 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''.]]
 
[[File:VEGFGFPCELL.png|400px|thumb|left|'''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.]]
 
 
</div>
 
<div style="display: block; height: 300pt;">
 
[[File:Sbnc.png|400px|thumb|left|'''Fig.20''' As results,there is no blue chromoprotein ''E. coli'' stick on the cell’s surface as there is no specific scFv displayed around the ''E. coli''.]]
 
[[File:VEGFAMILCPCELL.png|400px|thumb|left|'''Fig.21''' There are blue chromoprotein anti-VEGF ''E. coli'' stick on the cell’s surface as the anti-VEGF probes on ''E. coli'' successfully detect and bind with VEGF.]]
 
</div>
 
  
 
<h1>'''Modeling'''</h1>
 
<h1>'''Modeling'''</h1>
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 expression time by using the genetic algorithm (GA) 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 expression time.
 
<br>
 
<br>
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
+
By using the differential function which was derived from these optimum parameters which were calculated by GA can help us to simulate the optimum protein expression.
Compared with the simulated protein production rate of time, our experiment data quite fit the simulation.
+
When we have the simulated protein expression rate, the graph of protein expression versus time can be drawn.Thus, we can find the optimum protein expression time. However, the simulated protein expression curve is slower than the experimental curve by one hour. Therefore, to find the most exact optimum protein expression time, we infer that subtracting one hour of the optimum protein expression time would be correct.
 
<br>
 
<br>
 
<p style="font-size:120%">'''Co-transform'''</p>
 
<p style="font-size:120%">'''Co-transform'''</p>
 
<br>
 
<br>
[[File:Anti-VEGF-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.  
+
[[File:Anti-VEGF-GFP.jpg |900px|thumb|center|'''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.  
+
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.
 
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.]]
+
Thus, we can know that the E.Cotector can have maximum efficiency at this point.]]
  
[[File:VEGF-RFP.png|900px|thumb|left|'''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.]]  
+
[[File:VEGF-RFP.png|900px|thumb|left|'''Fig.12''' 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.]]  
  
[[File:VEGF-BFP.png|900px|thumb|left|'''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.]]  
+
[[File:VEGF-BFP.png|900px|thumb|left|'''Fig.13''' 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.]]  
  
 
<br>
 
<br>

Latest revision as of 11:08, 22 September 2015

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

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

2. Co-transform (Two plasmids)
(1) Parts

Fig.4 Pcons+RBS+Lpp-OmpA-N+anti-VEGF
Fig.5 Pcons+RBS+RFP+Ter
Fig.6 Pcons+RBS+GFP+Ter


(2) Cell staining

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.

Fig.7 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.8 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.9 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.10 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.


Modeling

In the modeling part, we discover optimum protein expression time by using the genetic algorithm (GA) in Matlab.
We want to characterize the actual kinetics of this Hill-function based model that accurately reflects protein expression time.
By using the differential function which was derived from these optimum parameters which were calculated by GA can help us to simulate the optimum protein expression. When we have the simulated protein expression rate, the graph of protein expression versus time can be drawn.Thus, we can find the optimum protein expression time. However, the simulated protein expression curve is slower than the experimental curve by one hour. Therefore, to find the most exact optimum protein expression time, we infer that subtracting one hour of the optimum protein expression time would be correct.

Co-transform


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 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.12 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.13 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
    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]