Part:BBa_K3279008
cenA gene fused with INP-N sequence
This part is a improvement of the previous part cenA gene (BBa_K118023). With the fusion with INP-N, the endoglucanase coded by cenA gene can rivet on the surface and achieve E.coli surface display[1].
Usage and Biology
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 333
Illegal BamHI site found at 733 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 75
Illegal NgoMIV site found at 408
Illegal AgeI site found at 1051
Illegal AgeI site found at 1414 - 1000COMPATIBLE WITH RFC[1000]
Characterization from iGEM19_CAU_China
This part is a improvement of the previous part cenA gene (BBa_K118023). With the fusion with INP-N, the endoglucanase coded by cenA gene can rivete on the surface and achieve E.coli surface display. We linked this part into a pET30a(+) backbone, then transformed this plasmid into BL21. We induced this recombinant E.coli strain overnight under the condition of 16℃ 0.08 mM. See Fig.1[1].
To confirm whether it worked or not, we first detected the presence of the target protein by immunofluorescence staining and compared the fluorescence pattern and compared it with a negative control where cellulases was not fused with INP. We attached the His-tag to the fusion protein, so that it could allow an anti-His-tag primary antibody to combine the fusion protein and then let a fluorescence secondary antibody recognize them. As the result, we could spot the fluorescence of GFP when INP-N was fused with cellulase. See Fig.2[1].
Then the effect of fusion on enzyme activity was detected by measuring the cellulose degradation ability using CMC-Na as substrate. See Fig.3[1].
| None |