Difference between revisions of "Part:BBa K3365059:Experience"
(→‎Applications of BBa_K3365059)
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To obtain MG1655-Δ<i>rpoZ</i>, MG1655 electroreceptive state was first prepared, and pKD46 plasmid was electransterred 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.
 
To obtain MG1655-Δ<i>rpoZ</i>, MG1655 electroreceptive state was first prepared, and pKD46 plasmid was electransterred 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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We then validated the knockout of MG1655, which knocked out the omega subunit, to prove that the knockout was successful. We first used Kana resistance for preliminary screening. Then we selected the colonies for PCR and verified them according to the band size and PCR products were sequenced for verification. Thus, we finally knock out <i>rpoZ</i> gene.
 
We then validated the knockout of MG1655, which knocked out the omega subunit, to prove that the knockout was successful. We first used Kana resistance for preliminary screening. Then we selected the colonies for PCR and verified them according to the band size and PCR products were sequenced for verification. Thus, we finally knock out <i>rpoZ</i> gene.
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[[File:The_result_of_PCR_for_verificating_the_selected_colonies.png]]
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<b>Fig:</b>The result of PCR for verificating the selected colonies
  
 
===User Reviews===
 
===User Reviews===

Revision as of 02:58, 26 October 2020


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Applications of BBa_K3365059

To obtain MG1655-ΔrpoZ, MG1655 electroreceptive state was first prepared, and pKD46 plasmid was electransterred 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

We then validated the knockout of MG1655, which knocked out the omega subunit, to prove that the knockout was successful. We first used Kana resistance for preliminary screening. Then we selected the colonies for PCR and verified them according to the band size and PCR products were sequenced for verification. Thus, we finally knock out rpoZ gene.

The result of PCR for verificating the selected colonies.png

Fig:The result of PCR for verificating the selected colonies

User Reviews

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