Difference between revisions of "Part:BBa K4377000"
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<partinfo>BBa_K4377000 short</partinfo> | <partinfo>BBa_K4377000 short</partinfo> | ||
| − | ''Drosophila melanogaster'' homeotic protein ultrabithorax(Ubx) coding region. It is said that protein can be produced as a kind of fiber by self-assembling with dityrosine bonds, and it has been proved to produce in Escherichia coli (''E. coli''). | + | ''Drosophila melanogaster'' homeotic protein ultrabithorax(Ubx) coding region. It is said that protein can be produced as a kind of fiber by self-assembling with dityrosine bonds, and it has been proved to produce in ''Escherichia coli'' (''E. coli''). |
===Design=== | ===Design=== | ||
In order to purify our protein after expression, we added 10x His tag at the N terminal of Ubx protein. The reason we add 10x His tag at N terminal but not C terminal is to amplify the production of Ubx protein. To improve performance, we performed codon harmonization. The optimized sequence of codons will be more suitable for expression by our engineered ''E. coli'' . | In order to purify our protein after expression, we added 10x His tag at the N terminal of Ubx protein. The reason we add 10x His tag at N terminal but not C terminal is to amplify the production of Ubx protein. To improve performance, we performed codon harmonization. The optimized sequence of codons will be more suitable for expression by our engineered ''E. coli'' . | ||
====Cloning result==== | ====Cloning result==== | ||
| − | We successfully amplified His | + | We successfully amplified His Ubx plasmid in ''E.coli'' Dh5α and transformed into ''E.coli'' BL21 for expression. |
| + | [[Image:BBa K4377000-digest check.jpg|400px]] | ||
====SDS-PAGE result==== | ====SDS-PAGE result==== | ||
| − | After expression, we conducted SDS PAGEs to verify whether the protein was produced. | + | After expression, we conducted SDS PAGEs to verify whether the protein was produced. |
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| + | [[Image:BBa K4377000-sds page.jpg|400px]] | ||
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==== Test ==== | ==== Test ==== | ||
As shown in the SDS PAGE result, the production was below expectation, also, we conducted functional tests, but there was no significant evidence that the protein is able to form dityrosine bonds. | As shown in the SDS PAGE result, the production was below expectation, also, we conducted functional tests, but there was no significant evidence that the protein is able to form dityrosine bonds. | ||
Latest revision as of 01:52, 14 October 2022
Ubx
Drosophila melanogaster homeotic protein ultrabithorax(Ubx) coding region. It is said that protein can be produced as a kind of fiber by self-assembling with dityrosine bonds, and it has been proved to produce in Escherichia coli (E. coli).
Design
In order to purify our protein after expression, we added 10x His tag at the N terminal of Ubx protein. The reason we add 10x His tag at N terminal but not C terminal is to amplify the production of Ubx protein. To improve performance, we performed codon harmonization. The optimized sequence of codons will be more suitable for expression by our engineered E. coli .
Cloning result
We successfully amplified His Ubx plasmid in E.coli Dh5α and transformed into E.coli BL21 for expression.
SDS-PAGE result
After expression, we conducted SDS PAGEs to verify whether the protein was produced.
Test
As shown in the SDS PAGE result, the production was below expectation, also, we conducted functional tests, but there was no significant evidence that the protein is able to form dityrosine bonds.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 103
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 316
- 1000COMPATIBLE WITH RFC[1000]