Part:BBa_K3888006:Design
UppE Downstream Flanking Region
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.
Figure 2: Deletion plasmid of UppE constructed according to the method mentioned above. UppE Upstream Flanking region is located from 1021bp to 2056bp.
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 513
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 513
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 513
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 513
Illegal NgoMIV site found at 172
Illegal NgoMIV site found at 318
Illegal NgoMIV site found at 442 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1001
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