Composite

Part:BBa_K3888998:Design

Designed by: Linhan Lei   Group: iGEM21_SJTang   (2021-10-20)
Revision as of 17:35, 20 October 2021 by RyanLei (Talk | contribs)


UppE 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 UppE knockout plasmid below. UppE Deletion Plasmid Map.png

Figure 2: Deletion plasmid of UppE constructed according to the method mentioned above.


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 694
    Illegal PstI site found at 1549
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 694
    Illegal PstI site found at 1549
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 694
    Illegal BglII site found at 333
    Illegal XhoI site found at 826
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 694
    Illegal PstI site found at 1549
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 694
    Illegal PstI site found at 1549
    Illegal NgoMIV site found at 38
    Illegal NgoMIV site found at 119
    Illegal NgoMIV site found at 157
    Illegal NgoMIV site found at 831
    Illegal NgoMIV site found at 943
    Illegal NgoMIV site found at 1208
    Illegal NgoMIV site found at 1354
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 538
    Illegal SapI.rc site found at 2037


Design Notes

Flanking region for 1k bp


Source

Rhodopseudomonas palustris CGA009

References

1. Fritts, Ryan K., et al. "A Rhizobiales-specific unipolar polysaccharide adhesin contributes to Rhodopseudomonas palustris biofilm formation across diverse photoheterotrophic conditions."Applied and environmental microbiology 83.4 (2017): e03035-16