Difference between revisions of "Part:BBa K1694045"
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<h1>'''Introduction:'''</h1>
 
<h1>'''Introduction:'''</h1>
 
[[File:HER2BFP.png|600px|thumb|center|'''Fig.1''' Pcons+B0034+Lpp-OmpA-N+scFv(anti-HER2)+B0030+BFP+J61048]]
 
[[File:HER2BFP.png|600px|thumb|center|'''Fig.1''' Pcons+B0034+Lpp-OmpA-N+scFv(anti-HER2)+B0030+BFP+J61048]]
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<p style="font-size:120%">'''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, which are Anti-VEGF+GFP, Anti-EGFR+RFP, Anti-HER2+BFP. 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.  
 
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, which are Anti-VEGF+GFP, Anti-EGFR+RFP, Anti-HER2+BFP. 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.  
  
 
<h1>'''Experiment'''</h1>
 
<h1>'''Experiment'''</h1>
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[[File:PROHBFPPCR.png|200px|thumb|left|'''Fig.3''' The PCR result of the Pcons+B0034+Lpp-OmpA-N+scFv(Anti-HER2)+B0030+BFP+J61048. The DNA sequence length is around 1900~2100 bp, so the PCR products should appear at 2100~2300 bp.]]
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'''1.Cloning'''
  
 
After assemble the DNA sequences from the basic parts, we recombined each Pcons+B0034+Lpp-OmpA-N+scFv(Anti-HER2)+B0030+BFP+J61048 gene to PSB1C3 backbones and conducted a PCR experiment to check the size of each of the parts. The DNA sequence length of the these parts are around 1900~2100 bp. In this PCR experiment, the PCR products size should be near at 2100~2300 bp. The Fig.3 showed the correct size of this part, and proved that we successful ligated the sequence onto an ideal backbone.
 
After assemble the DNA sequences from the basic parts, we recombined each Pcons+B0034+Lpp-OmpA-N+scFv(Anti-HER2)+B0030+BFP+J61048 gene to PSB1C3 backbones and conducted a PCR experiment to check the size of each of the parts. The DNA sequence length of the these parts are around 1900~2100 bp. In this PCR experiment, the PCR products size should be near at 2100~2300 bp. The Fig.3 showed the correct size of this part, and proved that we successful ligated the sequence onto an ideal backbone.
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[[File:PROHBFPPCR.png|200px|thumb|left|'''Fig.3''' The PCR result of the Pcons+B0034+Lpp-OmpA-N+scFv(Anti-HER2)+B0030+BFP+J61048. The DNA sequence length is around 1900~2100 bp, so the PCR products should appear at 2100~2300 bp.]]
  
 
[[File:PROHBFPsss.png|600px|thumb|center|'''Fig.4''' Pcons+B0034+Lpp-OmpA-N+scFv(anti-HER2)+B0030+BFP+J61048]]  
 
[[File:PROHBFPsss.png|600px|thumb|center|'''Fig.4''' Pcons+B0034+Lpp-OmpA-N+scFv(anti-HER2)+B0030+BFP+J61048]]  

Revision as of 20:53, 20 September 2015

Pcons+B0034+Lpp-OmpA-N+scFv(Anti-HER2)+B0030+BFP+J61048

Introduction:

Fig.1 Pcons+B0034+Lpp-OmpA-N+scFv(anti-HER2)+B0030+BFP+J61048

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, which are Anti-VEGF+GFP, Anti-EGFR+RFP, Anti-HER2+BFP. 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.

Experiment

1.Cloning

After assemble the DNA sequences from the basic parts, we recombined each Pcons+B0034+Lpp-OmpA-N+scFv(Anti-HER2)+B0030+BFP+J61048 gene to PSB1C3 backbones and conducted a PCR experiment to check the size of each of the parts. The DNA sequence length of the these parts are around 1900~2100 bp. In this PCR experiment, the PCR products size should be near at 2100~2300 bp. The Fig.3 showed the correct size of this part, and proved that we successful ligated the sequence onto an ideal backbone.

Fig.3 The PCR result of the Pcons+B0034+Lpp-OmpA-N+scFv(Anti-HER2)+B0030+BFP+J61048. The DNA sequence length is around 1900~2100 bp, so the PCR products should appear at 2100~2300 bp.
Fig.4 Pcons+B0034+Lpp-OmpA-N+scFv(anti-HER2)+B0030+BFP+J61048


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
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 741
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 451
    Illegal NgoMIV site found at 1831
  • 1000
    COMPATIBLE WITH RFC[1000]