Difference between revisions of "Part:BBa K3888999: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> | ||
| − | According to this model, we constructed | + | According to this model, we constructed Hups knockout plasmid below.<br> |
[[ File:HupS Deletion Plasmid Map.png]] | [[ File:HupS Deletion Plasmid Map.png]] | ||
===Figure 2: Deletion plasmid of HupS constructed according to the method mentioned above. The backbone we used is pK18mobsacB-Gm=== | ===Figure 2: Deletion plasmid of HupS constructed according to the method mentioned above. The backbone we used is pK18mobsacB-Gm=== | ||
Latest revision as of 10:07, 21 October 2021
HupS 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 Hups knockout plasmid below.
Figure 2: Deletion plasmid of HupS constructed according to the method mentioned above. The backbone we used is pK18mobsacB-Gm
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1521
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1521
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1521
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1521
Illegal NgoMIV site found at 121
Illegal NgoMIV site found at 258
Illegal NgoMIV site found at 690
Illegal NgoMIV site found at 1068 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 414
Design Notes
Our flanking region is about 1kbp in length for deletion.
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.