Difference between revisions of "Part:BBa K4377000"
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====Cloning result====
 
====Cloning result====
 
We successfully amplified His UBX plasmid in ''E.coli''  Dh5α and transformed into ''E.coli'' BL21 for expression.
 
We successfully amplified His UBX plasmid in ''E.coli''  Dh5α and transformed into ''E.coli'' BL21 for expression.
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[[Image:BBa K4377000-digest check.jpg|400px|Ubx plasmid extracted from E. coli BL21 digest check result. M - Marker. 1 - Ubx plasmid extracted from E. coli BL21 digest check result, backbone (pSB1A3, 2114bp) / insert (composite part K4377006, 1475bp).]]
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[[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:File:BBa K4377000-sds page.jpg|400px|Composite part K4377006 SDS-PAGE result. M - Marker. 1 - Control, pSB1A3 pure plasmid. 2 - Sample, Ubx plasmid, target band Ubx 42.56 kDa.]]
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[[Image:BBa K4377000-sds page.jpg|400px]]
  
  

Revision as of 00:01, 12 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. BBa K4377000-digest check.jpg

SDS-PAGE result

After expression, we conducted SDS PAGEs to verify whether the protein was produced. BBa K4377000-sds page.jpg


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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 103
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 316
  • 1000
    COMPATIBLE WITH RFC[1000]