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Since no record of the 3D structure of PncA is available in PDB, we turned to SWISS-MODEL for homology modeling to predict the 3D structure of PncA in Lactobacillus Plantarum L168.
Lactobacillus Plantarum L168, a strain we obtained from our PI, Xingyin Liu’s lab, has been proven to be able to alleviate social behavior deficits in animal models of autism. It carries the PncA gene itself and our aim is to amplify the gene, insert it into the vector, and transform the vector back to the L168 which enables the engineering bacteria to overexpress the PncA encoding protein. Thus, we designed primers, performed PCR, and ran gel to verify the successful amplification of the PncA gene.
As shown in the figure above,after PCR, the bands we obtained are near 600 bp, which was consistent with the target band (621 bp). This further indicated that we have successfully amplified the PncA gene. We then cut bands out of the agarose gel and purify the DNA samples. Using pLDHLH673 as the vector, we constructed the pLDHLH673-PncA plasmid expression system. To prove our construction is successful, we performed enzyme digestion verification on the plasmid, adding restriction endonuclease NdeI and XbaI for double enzyme digestion, and ran the agarose gel to verify.
As shown in the figure above, after enzyme digestion, the lower bands we obtained are near 600 bp, which was consistent with the target band (632 bp). The upper bands we obtained were higher than the marker and were near 5500bp, which was as we expected. This further indicated that have successfully constructed the pLDHLH673-PncA plasmid.