Composite

Part:BBa_K4800055

Designed by: Yifei Wu   Group: iGEM23_NJTech-China-B   (2023-10-11)
Revision as of 10:26, 12 October 2023 by ZQM 0907 (Talk | contribs)

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.

Document

Build:

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.

Test:

Figure 2. The 1,5-pentanediol production by the engineered strain of E. coli NT1003-P1.

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


Assembly Compatibility:
  • 10
    INCOMPATIBLE 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
  • 12
    INCOMPATIBLE 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
  • 21
    INCOMPATIBLE WITH RFC[21]
    Unknown
  • 23
    INCOMPATIBLE WITH RFC[23]
    Unknown
  • 25
    INCOMPATIBLE 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
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 9122
    Illegal SapI site found at 660


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