Coding

Part:BBa_K3888008:Design

Designed by: Xiaoxuan Cheng   Group: iGEM21_SJTang   (2021-10-01)


HupS Downstream Flanking Region
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.


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 520
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 520
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 520
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 520
    Illegal NgoMIV site found at 67
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Flanking region for 1k bp


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.