Difference between revisions of "Part:BBa K3888008:Experience"
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===Applications of BBa_K3888008===
 
===Applications of BBa_K3888008===
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[[File:Deletion Plasmid construction.png]]
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In order to achieve gene knockout, we used pK18mobsacB-Gm plasmid as a knockout vector. In addition, in order to achieve knockout, we selected about 1000bp sequences in the upstream and downstream of UppE and HupS respectively for design, amplified from the Rhodopseudomonas palustris CGA009 by PCR, and constructed knockout plasmids by the Gibson method (see the Design section for principles) ). The results of plasmid design and construction are as follows:
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===Figure 1: Illustration  of the general form of plasmid used to create marked knockout strains of R. palustris. ===
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[[File:HupS results 1.png]]
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The origin of replication (ori), origin of transfer (oriT) and sacB (blue) are part of the delivery plasmid. The green sequence is inserted into the multiple cloning site of the delivery plasmid. The left and right fragments (green) are sequences amplified from the R. palustris genome and used in overlap extension PCR to generate a recombinant product containing (a deletion and a restriction site）. The restriction site is used to introduce the resistance cassette (purple). This interrupts the gene even if no deletion is present, and allows selection of successful transformants. <br>
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===Figure 1. Plasmid Design and Agarose Gel Result. Left: UppE Plasmid Design. Middle: HupS Plasmid Design. Right: Agarose gel result (0.8%).===
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According to this model, we constructed UppE knockout plasmid below.<br>
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After the plasmid construction is completed, we use the method of electrotransformation to transfer the plasmid into the cell. Afterwards, colonies in which the plasmid was successfully integrated into the chromosome were screened by the gentamicin resistance gene on the vector. After that, the medium containing 10% sucrose was used for secondary screening to facilitate the excision of the inserted plasmid sequence in the double exchange event. Finally, the selected individuals are amplified and cultured, and the genome is extracted and verified by PCR. In order to advance the progress of the experiment, the first round of knockout experiments were carried out separately, and the knockout results are as follows:
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[[ File:HupS Deletion Plasmid Map.png]]
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[[File:HupS results 2.png]]
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===Figure 2: Deletion plasmid of HupS constructed according to the method mentioned above.===
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===Figure 2. Gene knockout design and result. Left: HupS Test Design. Right:HupS Test Result. The results show that the deletion of HupS gene was successful.===
  
 
===User Reviews===
 
===User Reviews===

Revision as of 19:46, 20 October 2021


Applications of BBa_K3888008

In order to achieve gene knockout, we used pK18mobsacB-Gm plasmid as a knockout vector. In addition, in order to achieve knockout, we selected about 1000bp sequences in the upstream and downstream of UppE and HupS respectively for design, amplified from the Rhodopseudomonas palustris CGA009 by PCR, and constructed knockout plasmids by the Gibson method (see the Design section for principles) ). The results of plasmid design and construction are as follows: HupS results 1.png

Figure 1. Plasmid Design and Agarose Gel Result. Left: UppE Plasmid Design. Middle: HupS Plasmid Design. Right: Agarose gel result (0.8%).

After the plasmid construction is completed, we use the method of electrotransformation to transfer the plasmid into the cell. Afterwards, colonies in which the plasmid was successfully integrated into the chromosome were screened by the gentamicin resistance gene on the vector. After that, the medium containing 10% sucrose was used for secondary screening to facilitate the excision of the inserted plasmid sequence in the double exchange event. Finally, the selected individuals are amplified and cultured, and the genome is extracted and verified by PCR. In order to advance the progress of the experiment, the first round of knockout experiments were carried out separately, and the knockout results are as follows: HupS results 2.png

Figure 2. Gene knockout design and result. Left: HupS Test Design. Right:HupS Test Result. The results show that the deletion of HupS gene was successful.

User Reviews

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Reference: Rey, Federico E., Yasuhiro Oda, and Caroline S. Harwood. "Regulation of uptake hydrogenase and effects of hydrogen utilization on gene expression in Rhodopseudomonas palustris." Journal of bacteriology 188.17 (2006): 6143-6152. UNIQfa4153db5dd900a3-partinfo-00000001-QINU