Difference between revisions of "Part:BBa K4800055"

Revision as of 10:20, 12 October 2023

pRSFDuet-sfp-MmCAR-YahK

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.

Build:

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 < a href=' '>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.

Test:

The yield of 1,5-PDO produced by the engineered strain NT1003-P2 was determined by the HPLC analysis. After a total fermentation period of 84 h, 8.09 mM (0.843g/L) 1,5-PDO was produced by the engineered strain E. coli NT1003-P2, which is 3.5-fold higher than that in E. coli NT1003-P1.

Sequence and Features