Difference between revisions of "Part:BBa K3888998:Design"
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===Figure 1: Illustration of the general form of plasmid used to create marked knockout strains of R. palustris. === | ===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. <br> | 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. <br> | ||
Revision as of 17:34, 20 October 2021
UppE Knockout
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.
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 694
Illegal PstI site found at 1549 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 694
Illegal PstI site found at 1549 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 694
Illegal BglII site found at 333
Illegal XhoI site found at 826 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 694
Illegal PstI site found at 1549 - 25INCOMPATIBLE 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 - 1000INCOMPATIBLE 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