Difference between revisions of "Part:BBa K3365059"
 
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This part is designed to knock out genome <i>rpoZ</i>, which codes for the Omega submit of RNA polymerase. We added 40 base pairs homologous arm at both end of Kanamycin resistant gene in order to replace <i>rpoZ</i>.  
 
This part is designed to knock out genome <i>rpoZ</i>, which codes for the Omega submit of RNA polymerase. We added 40 base pairs homologous arm at both end of Kanamycin resistant gene in order to replace <i>rpoZ</i>.  
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To obtain MG1655-Δ<i>rpoZ</i>, MG1655 electroreceptive state was first prepared, and pKD46 plasmid was electrotransfromed into MG1655. At the same time, we used pET28a plasmid as the template to obtain the Kana-homo fragment with the homologous arms on both ends of the <i>rpoZ</i> gene by PCR. Then, L-arabinose was added to induce pKD46 gene expression of Exo、Beta、Gam protein, and Kana-homo fragment was electransterred in to knock out <i>rpoZ</i> gene.
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[[File:PCR_product_of_Kana-homo_fragment_with_the_homologous_arms.png]]
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Then, L-arabinose was added to induce pKD46 gene expression of Exo、Beta、Gam protein, and Kana-homo fragment was electrotransformed to knock out <i>rpoZ</i> gene.
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The sequencing result showed that the knockout was successful. We first used Kana resistance for preliminary screening. We selected the colonies for PCR and applied a gel verification on it.  The gel graph shown below, again, showed that the knockout was a success.
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[[File:The_result_of_PCR_for_verificating_the_selected_colonies.png]]
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<b>Fig:</b>Verification of <i>rpoZ</i> deletion through colony PCR
  
 
[[File:Sequencing_result_firstly.png]]
 
[[File:Sequencing_result_firstly.png]]

Latest revision as of 12:51, 27 October 2020


kanamycin resistant gene with homologous arms

This part is designed to knock out genome rpoZ, which codes for the Omega submit of RNA polymerase. We added 40 base pairs homologous arm at both end of Kanamycin resistant gene in order to replace rpoZ.

To obtain MG1655-ΔrpoZ, MG1655 electroreceptive state was first prepared, and pKD46 plasmid was electrotransfromed into MG1655. At the same time, we used pET28a plasmid as the template to obtain the Kana-homo fragment with the homologous arms on both ends of the rpoZ gene by PCR. Then, L-arabinose was added to induce pKD46 gene expression of Exo、Beta、Gam protein, and Kana-homo fragment was electransterred in to knock out rpoZ gene.

PCR product of Kana-homo fragment with the homologous arms.png

Then, L-arabinose was added to induce pKD46 gene expression of Exo、Beta、Gam protein, and Kana-homo fragment was electrotransformed to knock out rpoZ gene. The sequencing result showed that the knockout was successful. We first used Kana resistance for preliminary screening. We selected the colonies for PCR and applied a gel verification on it. The gel graph shown below, again, showed that the knockout was a success.

The result of PCR for verificating the selected colonies.png

Fig:Verification of rpoZ deletion through colony PCR

Sequencing result firstly.png

Sequencing result secondly.png

Fig:According to the sequencing result, the new sequence, which was rpoZ before, is no longer similar to rpoZ any more, and it is nearly the same sequence as the KanR. This shows that we replace the rpoZ with KanR successfully.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]