Difference between revisions of "Part:BBa K3279008"
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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]. | 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]. | ||
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| + | ===Usage and Biology=== | ||
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| + | <span class='h3bb'>Sequence and Features</span> | ||
| + | <partinfo>BBa_K3279008 SequenceAndFeatures</partinfo> | ||
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===Characterization from iGEM19_CAU_China=== | ===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. | 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]. | 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]. | ||
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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]. | 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]. | ||
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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]. | 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]. | ||
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Revision as of 15:54, 15 October 2019
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].