Composite

Part:BBa_K2207015:Design

Designed by: Junming Qian   Group: iGEM17_ZJU-China   (2017-10-26)


PhlBD enzyme


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 1582
    Illegal XbaI site found at 3794
    Illegal PstI site found at 776
    Illegal PstI site found at 3806
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 776
    Illegal PstI site found at 3806
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1494
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 1582
    Illegal XbaI site found at 3794
    Illegal PstI site found at 776
    Illegal PstI site found at 3806
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 1582
    Illegal XbaI site found at 3794
    Illegal PstI site found at 776
    Illegal PstI site found at 3806
    Illegal NgoMIV site found at 1298
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 486
    Illegal BsaI.rc site found at 2279
    Illegal SapI site found at 168


Design Notes

We used the F2A sequenece（gtgaaacagactttgaattttgaccttctcaagttggcgggagacgtggagtccaaccctggacct）to link the phlB with phlD, and then combined them to eGFP to construct a fusion protein so that the two genes can be transcribed as a single mRNA and generating two independent gene products, and meanwhile we can identify whether the proteins are expressed by detecting the fluorescent.


Source

We successfully cloned the phlBD via colony PCR from Pseudomonas fluorescens 2P24, which is a gift from Prof. Liqun Zhang of China Agriculture University.


References

Jin H K, Lee S R, Li L H, et al. High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and Mice[J]. Plos One, 2011, 6(4):e18556.