Difference between revisions of "Part:BBa K1991010"
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=== GENE EXPRESSION SYSTEM === | === GENE EXPRESSION SYSTEM === | ||
| − | <p>The AOX gene were cloned onto pSB1C3 driven by a constitutive promoter (BBa_J23101) and RBS (BBa_B0034) 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 | + | <p>The AOX gene were cloned onto pSB1C3 driven by a constitutive promoter ([https://parts.igem.org/Part:BBa_J23101 BBa_J23101]) and RBS ([https://parts.igem.org/Part:BBa_B0034 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 ([https://parts.igem.org/Part:BBa_K1694035 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.</p> |
[[File:2016Mingdao_GFP123.jpg|300px|thumb|left]] | [[File:2016Mingdao_GFP123.jpg|300px|thumb|left]] | ||
Latest revision as of 05:06, 20 October 2016
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NotI site found at 1220 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 497
Illegal XhoI site found at 1229 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 452
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1143