Difference between revisions of "Part:BBa K1991007"
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__NOTOC__
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<partinfo>BBa_K1991007 short</partinfo>
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A constitutive promoter ([https://parts.igem.org/Part:BBa_J23101 BBa_J23101]) and RBS ([https://parts.igem.org/Part:BBa_B0034 BBa_B0034]) were added in front of Lpp-OmpA-BamHI ([https://parts.igem.org/Part:BBa_K1991004 BBa_K1991004]) to facilitate fusion protein gene expression.
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<p>Existing part from NCTU-Formosa in 2015:</p>
 
<p>Existing part from NCTU-Formosa in 2015:</p>
 
<ul><li>[https://parts.igem.org/Part:BBa_K1694002 BBa_K1694002]: Lpp-OmpA-NcoI/pSB1C3</li></ul>
 
<ul><li>[https://parts.igem.org/Part:BBa_K1694002 BBa_K1694002]: Lpp-OmpA-NcoI/pSB1C3</li></ul>
 
<p>Improved part by Mingdao in 2016: </p>
 
<p>Improved part by Mingdao in 2016: </p>
 
<ul>
 
<ul>
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     <li>[https://parts.igem.org/Part:BBa_K1991004 BBa_K1991004]: Lpp-OmpA-BamHI/pSB1C3<</li>
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     <li>[https://parts.igem.org/Part:BBa_K1991004 BBa_K1991004]: Lpp-OmpA-BamHI/pSB1C3</li>
 
<li>[https://parts.igem.org/Part:BBa_K1991007 BBa_K1991007]: Pcons-RBS-LO-BamHI/pSB1C3</li>
 
<li>[https://parts.igem.org/Part:BBa_K1991007 BBa_K1991007]: Pcons-RBS-LO-BamHI/pSB1C3</li>
 
</ul>
 
</ul>
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  <p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
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== Lpp-OmpA Cell Surface Display System ==
 
     <p>Lpp and OmpA are outer membrane proteins of E. coli. Lpp-OmpA (LO) hybrid can direct heterologous proteins to bacterial cell surface. In 2015, NCTU-Formosa used it to display scFv (single chain fragment variable) antibodies on the surface of E. coli. They found that a fusion protein cannot be possible to be created under the standard BioBrick assembly rule, that is EcoRI(E)-XbaI(X)-GENE-SpeI(S)-PstI(P). The A part of EX-LO-SP and the B part of EX-scFV-SP, for example, are connected by cutting and ligation of SpeI plus PstI for the A part and XbaI plus PstI for the B part. The SCAR generated by XbaI/SpeI (ACTAGA) will form a stop codon just in front of the ATG start codon of the scFV protein of the B part. This situation has been officially mentioned by the [http://openwetware.org/wiki/The_BioBricks_Foundation:BBFRFC15#Limitations_of_Standard_Assembly BioBrick standard assembly]. </p>
 
     <p>Lpp and OmpA are outer membrane proteins of E. coli. Lpp-OmpA (LO) hybrid can direct heterologous proteins to bacterial cell surface. In 2015, NCTU-Formosa used it to display scFv (single chain fragment variable) antibodies on the surface of E. coli. They found that a fusion protein cannot be possible to be created under the standard BioBrick assembly rule, that is EcoRI(E)-XbaI(X)-GENE-SpeI(S)-PstI(P). The A part of EX-LO-SP and the B part of EX-scFV-SP, for example, are connected by cutting and ligation of SpeI plus PstI for the A part and XbaI plus PstI for the B part. The SCAR generated by XbaI/SpeI (ACTAGA) will form a stop codon just in front of the ATG start codon of the scFV protein of the B part. This situation has been officially mentioned by the [http://openwetware.org/wiki/The_BioBricks_Foundation:BBFRFC15#Limitations_of_Standard_Assembly BioBrick standard assembly]. </p>
 
     <div class="propic3">
 
     <div class="propic3">
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<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
 
     [[File:T--Mingdao--EX1.jpg|600px|thumb|left]]
 
     [[File:T--Mingdao--EX1.jpg|600px|thumb|left]]
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         <p>Figure 1: Limitation of cloning a fusion protein by standard biobrick assembly    </p>
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<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
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         <p>[https://static.igem.org/mediawiki/2016/2/2c/T--Mingdao--EX1.jpg Figure 1]: Limitation of cloning a fusion protein by standard biobrick assembly    </p>
 
     </div>
 
     </div>
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<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
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<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
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== NCTU-FORMOSA 2015 ==
 
<p>Therefore, in 2015, NCTU-Formosa created a novel part ([https://parts.igem.org/Part:BBa_K1694002 BBa_K1694002]) putting NcoI site between the LO part and SpeI site. However, when considering cloning, we found that an extra NcoI site is present on Cm resistance gene making it difficult be a vector for gene cloning.</p>
 
<p>Therefore, in 2015, NCTU-Formosa created a novel part ([https://parts.igem.org/Part:BBa_K1694002 BBa_K1694002]) putting NcoI site between the LO part and SpeI site. However, when considering cloning, we found that an extra NcoI site is present on Cm resistance gene making it difficult be a vector for gene cloning.</p>
 +
<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
 +
== MINGDAO 2016 ==
 
     <p>In 2016, Mingdao improved the part by replacing NcoI site with BamHI site ([https://parts.igem.org/Part:BBa_K1991004 BBa_K1991004]). Also, we’ve confirmed and [http://2016.igem.org/Team:Mingdao/Proof prove] the function of LO directing a fusion protein to the cell surface with enzyme activity in our project. </p>
 
     <p>In 2016, Mingdao improved the part by replacing NcoI site with BamHI site ([https://parts.igem.org/Part:BBa_K1991004 BBa_K1991004]). Also, we’ve confirmed and [http://2016.igem.org/Team:Mingdao/Proof prove] the function of LO directing a fusion protein to the cell surface with enzyme activity in our project. </p>
  
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         [[File:T--Mingdao--EX3.jpg|600px|thumb|left]]  
 
         [[File:T--Mingdao--EX3.jpg|600px|thumb|left]]  
  
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       <p>Figure 2: Alternative methods of cloning a fusion protein on Biobrick parts designed and created by NCTU-FORMOSA in 2015 and MINGDAO in 2016</p>
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<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
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<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
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       <p>Figure [https://static.igem.org/mediawiki/2016/7/7f/T--Mingdao--EX2.jpg 2-1], [https://static.igem.org/mediawiki/2016/1/1d/T--Mingdao--EX3.jpg 2-2]: Alternative methods of cloning a fusion protein on Biobrick parts designed and created by NCTU-FORMOSA in 2015 and MINGDAO in 2016</p>
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<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
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<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
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<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
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<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
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<!-- Add more about the biology of this part here
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1991007 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K1991007 parameters</partinfo>
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<!-- -->

Revision as of 17:43, 19 October 2016

Pcons-RBS-LO-BamHI

A constitutive promoter (BBa_J23101) and RBS (BBa_B0034) were added in front of Lpp-OmpA-BamHI (BBa_K1991004) to facilitate fusion protein gene expression.


Existing part from NCTU-Formosa in 2015:

Improved part by Mingdao in 2016:

 

Lpp-OmpA Cell Surface Display System

Lpp and OmpA are outer membrane proteins of E. coli. Lpp-OmpA (LO) hybrid can direct heterologous proteins to bacterial cell surface. In 2015, NCTU-Formosa used it to display scFv (single chain fragment variable) antibodies on the surface of E. coli. They found that a fusion protein cannot be possible to be created under the standard BioBrick assembly rule, that is EcoRI(E)-XbaI(X)-GENE-SpeI(S)-PstI(P). The A part of EX-LO-SP and the B part of EX-scFV-SP, for example, are connected by cutting and ligation of SpeI plus PstI for the A part and XbaI plus PstI for the B part. The SCAR generated by XbaI/SpeI (ACTAGA) will form a stop codon just in front of the ATG start codon of the scFV protein of the B part. This situation has been officially mentioned by the [http://openwetware.org/wiki/The_BioBricks_Foundation:BBFRFC15#Limitations_of_Standard_Assembly BioBrick standard assembly].

 

File:T--Mingdao--EX1.jpg
600px

 

Figure 1: Limitation of cloning a fusion protein by standard biobrick assembly

 

 

NCTU-FORMOSA 2015

Therefore, in 2015, NCTU-Formosa created a novel part (BBa_K1694002) putting NcoI site between the LO part and SpeI site. However, when considering cloning, we found that an extra NcoI site is present on Cm resistance gene making it difficult be a vector for gene cloning.

 

MINGDAO 2016

In 2016, Mingdao improved the part by replacing NcoI site with BamHI site (BBa_K1991004). Also, we’ve confirmed and [http://2016.igem.org/Team:Mingdao/Proof prove] the function of LO directing a fusion protein to the cell surface with enzyme activity in our project.

File:T--Mingdao--EX2.jpg
600px
File:T--Mingdao--EX3.jpg
600px

 

 

Figure 2-1, 2-2: Alternative methods of cloning a fusion protein on Biobrick parts designed and created by NCTU-FORMOSA in 2015 and MINGDAO in 2016

 

 

 

 

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 BamHI site found at 497
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 452
  • 1000
    COMPATIBLE WITH RFC[1000]