Part:BBa_K1991010

Pcons-RBS-LO-GFP-His

GENE EXPRESSION SYSTEM

The AOX gene were cloned onto pSB1C3 driven by a constitutive promoter (BBa_J23101) and RBS (BBa_K1694035) and fused with bacterial outer membrane proteins, Lpp-OmpA (LO) to display protein on the cell surface of E. coli. To test whether the gene expression system works, we cloned a reporter gene (GFP) in the same context (i.e., Pcons-RBS-LO-GFP/pSB1C3). And the gene expression level was compared to Pcons-RBS-GFP/pSB1C3 (BBa_K1694035) got from NCTU-Formosa, which has the same promoter and RBS but without LO. The clones of E. coli DH5 were cultured in LB media supplemented with 34 ug/ml chloramphenicol at 37°C overnight. Because overnight cultured LB medium has a background level of fluorescence, the bacterial GFP was measured in the PBS buffer (Enzyme Microb Technol. 2001). As Figure 1 showed, the GFP expression was extremely high in E. coli. LO-GFP fusion protein expression was observed at low but significant level compared to wild-type E. coli or E. coli expressing LO outer membrane proteins.

 

 

Figure: Gene expression analysis. GFP gene expression was read at Ex/Em = 488/528 nm in BioTek Microplate Spectrophotometer. Lane 1: wild-type E. coli as a mock control; Lane 2: GFP expression in E. coli (BBa_K1694035); Lane 3: LO outer membrane protein expression (BBa_K1991007) in E. coli; Lane 4: LO-GFP fusion protein expression (BBa_K1991010) in E. coli.

 

 

 

 

 

 

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
  Illegal NotI site found at 1220
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 497
  Illegal XhoI site found at 1229
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 452
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1143