Part:BBa_K1991007

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:

• BBa_K1694002: Lpp-OmpA-NcoI/pSB1C3

Improved part by Mingdao in 2016:

• BBa_K1991004: Lpp-OmpA-BamHI/pSB1C3
• BBa_K1991007: Pcons-RBS-LO-BamHI/pSB1C3

 

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].

 

 

 

 

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.

 

 

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
  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]