Composite

Part:BBa_K3888998

Designed by: Linhan Lei   Group: iGEM21_SJTang   (2021-10-20)


UppE Knockout

UppE Knockout
This composite part is used for UppE knockout. Upp encodes part of the unipolar polysaccharide (UPP) and is has been showed that UPP is important for adhesion when light and organic substrates are used for growth for R. palustris CGA009. UppE(RPA2750) is located on 3120549..3122105 on the complete genome of R. palustris. This part consists of two basic parts, which are UppE upstream flanking region (BBa_K3888005) and a UppE downstream flanking region(BBa_K3888006), both have a length of about 1000bp.
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.

Sequence and Features


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


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