Composite

Part:BBa_K3888999:Design

Designed by: Xiaoxuan Cheng   Group: iGEM21_SJTang   (2021-10-20)


HupS knockout
Deletion Plasmid construction.png

Figure 1: Illustration of the general form of plasmid used to create marked knockout strains of R. palustris.

The origin of replication (ori), origin of transfer (oriT) and sacB (blue) are part of the delivery plasmid. The green sequence is inserted into the multiple cloning site of the delivery plasmid. The left and right fragments (green) are sequences amplified from the R. palustris genome and used in overlap extension PCR to generate a recombinant product containing (a deletion and a restriction site）. The restriction site is used to introduce the resistance cassette (purple). This interrupts the gene even if no deletion is present, and allows selection of successful transformants.
According to this model, we constructed Hups knockout plasmid below.
HupS Deletion Plasmid Map.png

Figure 2: Deletion plasmid of HupS constructed according to the method mentioned above. The backbone we used is pK18mobsacB-Gm


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1521
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1521
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1521
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1521
    Illegal NgoMIV site found at 121
    Illegal NgoMIV site found at 258
    Illegal NgoMIV site found at 690
    Illegal NgoMIV site found at 1068
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 414


Design Notes

Our flanking region is about 1kbp in length for deletion.


Source

Rhodopseudomonas palustris CGA009

References

Rey, Federico E., Yasuhiro Oda, and Caroline S. Harwood. "Regulation of uptake hydrogenase and effects of hydrogen utilization on gene expression in Rhodopseudomonas palustris." Journal of bacteriology 188.17 (2006): 6143-6152.