Difference between revisions of "Part:BBa K4377000:Design"

(→‎Design Notes)
(→‎Design Notes)
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According to Escherichia coli codon preference.
 
According to Escherichia coli codon preference.
  
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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
+
<p>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</p>
 +
 
 +
<h4>Cloning result</h4>
 +
<p>We successfully amplified His UBX plasmid in E.coli  Dh5α and transformed into E.coli BL21 for expression.</p>
 +
<figure></figure>
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 +
<h4>SDS-PAGE result</h4>
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<p>After expression, we conducted SDS PAGEs to verify whether the protein was produced.( result in Figure 3.)</p>
 +
<figure></figure>
 +
 
 +
<h4>Test</h4>
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<p>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.</p>
  
 
===Source===
 
===Source===

Revision as of 19:42, 11 October 2022


Ubx


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]


Design Notes

According to Escherichia coli codon preference.

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.

<figure></figure>

SDS-PAGE result

After expression, we conducted SDS PAGEs to verify whether the protein was produced.( result in Figure 3.)

<figure></figure>

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.

Source

Drosophila melanogaster, sequence found through Uniprot https://www.uniprot.org/uniprotkb/P83949/entry

References