Part:BBa_K3888998:Experience

Applications of BBa_K3888005

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:

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:

Figure 2. Gene knockout design.

The knockout screening of UppE was not successful. In the end, we failed to complete the knockout of UppE.

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