Part:BBa_K4800055

_NOTOC__ YcjQ sgRNA

The plasmid of pRSFDuet-sfp-MmCAR-YahK were constructed to obtain the functional modules for 1,5-pentanediol.We co-transformed the plasmid of pTrc99a-davB-davA-GabT and pRSFDuet-sfp-MmCAR-YahK into a lysine producing strain E. coli NT1003 to produce 1,5-PDO from glucose.

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Figure 1. The schematic of plasmid pRSFDuet-sfp-MmCAR-YahK.

The genes of 1,5-PDO synthesis module including MmCAR and YahK were subcloned into the plasmid pACYCDuet together under the control of trc promoter to yield plasmid pACYCDuet-sfp-MmCAR-YahK. In detail, based on the sequence of an existing part of BBa_K1655000 , it was codon-optimized and synthesized to obtain the fragment of sfp-MmCAR, which was then ligated into the pACYCDuet plasmid under the control of trc promoter. The Ptrc-YahK fragment was amplified with plasmid pTrc99a-YahK as the template, and In-Fusion cloning was then used to ligate into the above vectors to yield plasmid pACYCDuet-sfp-MmCAR-YahK. The plasmid were transformed into E. coli Trans-T1, and corrected colonies were verified by PCR, and sequencing.

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Figure 2.