Difference between revisions of "Part:BBa K1991004"
 
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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>
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        <p>Figure 1: Limitation of cloning a fusion protein by standard biobrick assembly    </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>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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Latest revision as of 08:29, 20 October 2016

Lpp-OmpA-BamHI

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].

 

2016Mingdao EX1.jpg

 

 

 

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.

2016Mingdao EX2.jpg
2016Mingdao EX3.jpg

 

 

Figure 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
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 436
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 391
  • 1000
    COMPATIBLE WITH RFC[1000]