Difference between revisions of "Part:BBa K1694015"
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<h1>'''Introduction:'''</h1> | <h1>'''Introduction:'''</h1> | ||
− | [[File: | + | [[File:H11.png|200px|thumb|right|'''Fig.1''' OmpA-N-scFv (anti-HER2)]] |
− | To display the | + | To display the <html><a href="https://parts.igem.org/Part:BBa_K1694005"> scFv</a></html> outside the ''E. coli'', we used <html><a href="https://parts.igem.org/Part:BBa_K1694002">Lipoprotein-Outer membrane protein A (Lpp-OmpA)</a></html>. According to the paper reference, we chose the first 9 amino acid of Lpp and the 46~159 amino acid of OmpA. |
<br> | <br> | ||
− | In order to change | + | In order to easily change various scFv DNA sequence, we added a ''NcoI'' restriction site between OmpA and scFv. When designing XbaI-SpeI restriction site between Lpp-OmpA and scFv, it will cause a mixed site. Therefore, the NcoI restriction site rather than the EX-SP restriction site was designed. |
+ | |||
+ | <br> | ||
+ | The Fig.2 showed how we design ''NcoI'' restriction site. First, we digest Lpp-OmpA part with EcoRI and NcoI, scFv part with NcoI PstI. Then we ligated them together. | ||
+ | |||
+ | <br> | ||
+ | <div style="display: block; height: 250pt;"> | ||
+ | [[File:131415.png|600px|thumb|left|'''Fig.2''' The combination of OmpA-N-scFv]] | ||
+ | [[File:ompascfv.png|250px|thumb|left|'''Fig.3''' OmpA-N-scFv]] | ||
+ | </div> | ||
+ | <br><br> | ||
+ | |||
+ | <html> | ||
+ | Introduction of basic parts: | ||
+ | <br> | ||
+ | <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1694015">OmpA-Anti-HER2 </a><br> | ||
+ | </html> | ||
+ | |||
+ | |||
+ | |||
<h1>'''Experiment:'''</h1> | <h1>'''Experiment:'''</h1> | ||
− | After receiving the DNA sequences from the gene synthesis company, we recombined each OmpA-N-scFv gene to | + | |
+ | [[File:OH.png|200px|thumb|left|'''Fig.4''' The PCR result of the Lpp-OmpA-N+scFv. The DNA sequence length is 1026 bp, so the PCR products should appear at 1200~1400 bp.]] | ||
+ | |||
+ | After receiving the DNA sequences from the gene synthesis company, we recombined each OmpA-N-scFv gene to pSB1C3 backbones and conducted a PCR experiment to check the size of each of the OmpA-N-scFvs. The length of OmpA-N-scFv DNA sequence is around 1000~1200 bp. In this PCR experiment, the OmpA-N-scFv products size should be near at 1200~1400 bp. The Fig.4 showed the correct size of the scFv, and proved that we successfully ligated the scFv sequence onto an ideal backbone. | ||
+ | |||
+ | [[File:OmpA-H.png|600px|thumb|center|'''Fig.5''' OmpA-N-scFv (anti-HER2)]] | ||
+ | |||
+ | |||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
===Usage and Biology=== | ===Usage and Biology=== |
Latest revision as of 07:43, 22 September 2015
OmpA-scFv(Anti-HER2)
Introduction:
To display the scFv outside the E. coli, we used Lipoprotein-Outer membrane protein A (Lpp-OmpA). According to the paper reference, we chose the first 9 amino acid of Lpp and the 46~159 amino acid of OmpA.
In order to easily change various scFv DNA sequence, we added a NcoI restriction site between OmpA and scFv. When designing XbaI-SpeI restriction site between Lpp-OmpA and scFv, it will cause a mixed site. Therefore, the NcoI restriction site rather than the EX-SP restriction site was designed.
The Fig.2 showed how we design NcoI restriction site. First, we digest Lpp-OmpA part with EcoRI and NcoI, scFv part with NcoI PstI. Then we ligated them together.
Introduction of basic parts:
OmpA-Anti-HER2
Experiment:
After receiving the DNA sequences from the gene synthesis company, we recombined each OmpA-N-scFv gene to pSB1C3 backbones and conducted a PCR experiment to check the size of each of the OmpA-N-scFvs. The length of OmpA-N-scFv DNA sequence is around 1000~1200 bp. In this PCR experiment, the OmpA-N-scFv products size should be near at 1200~1400 bp. The Fig.4 showed the correct size of the scFv, and proved that we successfully ligated the scFv sequence onto an ideal backbone.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 678
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 388
- 1000COMPATIBLE WITH RFC[1000]