Difference between revisions of "Part:BBa K3279008"

Revision as of 16:31, 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

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.

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.

Then the effect of fusion on enzyme activity was detected by measuring the cellulose degradation ability using CMC-Na as substrate. See Fig.3. Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 333
Illegal BamHI site found at 733
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE 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
1000
COMPATIBLE WITH RFC[1000]

@@ Line 7: / Line 7: @@
 ===Usage and Biology===
 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.
-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.
-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.
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 <span class='h3bb'>Sequence and Features</span>