Part:BBa_K4800055
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 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
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 4145
Illegal XbaI site found at 1415
Illegal PstI site found at 4580
Illegal PstI site found at 4607
Illegal PstI site found at 5876
Illegal PstI site found at 5903
Illegal PstI site found at 6296
Illegal PstI site found at 6792
Illegal PstI site found at 7436 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 4145
Illegal NheI site found at 7704
Illegal PstI site found at 4580
Illegal PstI site found at 4607
Illegal PstI site found at 5876
Illegal PstI site found at 5903
Illegal PstI site found at 6296
Illegal PstI site found at 6792
Illegal PstI site found at 7436 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 4145
Illegal BglII site found at 5134
Illegal BglII site found at 5760
Illegal BglII site found at 6036
Illegal BglII site found at 6727
Illegal BamHI site found at 4117
Illegal XhoI site found at 7676 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 4145
Illegal XbaI site found at 1415
Illegal PstI site found at 4580
Illegal PstI site found at 4607
Illegal PstI site found at 5876
Illegal PstI site found at 5903
Illegal PstI site found at 6296
Illegal PstI site found at 6792
Illegal PstI site found at 7436 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 4145
Illegal XbaI site found at 1415
Illegal PstI site found at 4580
Illegal PstI site found at 4607
Illegal PstI site found at 5876
Illegal PstI site found at 5903
Illegal PstI site found at 6296
Illegal PstI site found at 6792
Illegal PstI site found at 7436
Illegal NgoMIV site found at 6404
Illegal NgoMIV site found at 6466
Illegal NgoMIV site found at 6683
Illegal NgoMIV site found at 7130
Illegal AgeI site found at 4310
Illegal AgeI site found at 5321
Illegal AgeI site found at 5618
Illegal AgeI site found at 6503
Illegal AgeI site found at 6512
Illegal AgeI site found at 7187
Illegal AgeI site found at 9049 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 9122
Illegal SapI site found at 660
None |