Difference between revisions of "Part:BBa K3888005"

Revision as of 14:20, 20 October 2021

UppE Upstream Flanking Region

UppE Upstream Flanking Region
This basic part is the upstream flanking region of UppE for R. palustris Together with UppE downstream flanking region(BBa_K3888006), it forms UppE Knockout(BBa_K3888012).
Upp encodes part of the unipolar polysaccharide (UPP) and it has been proved that UPP is important for adhesion when light and organic substrates are used for growth for R. palustris UppE(RPA2750) is located on 3120549..3122105 on the complete genome 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.
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.

Sequence and Features UppE upstream flanking region is located from 1bp to 1020bp, which has a length of 1020bp.

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 694
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 694
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
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 694
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
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 538

@@ Line 8: / Line 8: @@
 [[File: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.
+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 UppE knockout plasmid below.
 [[File:UppE Deletion Plasmid Map.png]]
@@ Line 18: / Line 18: @@
 <!-- -->
 <span class='h3bb'>Sequence and Features</span>
+UppE upstream flanking region is located from 1bp to 1020bp, which has a length of 1020bp.
 <partinfo>BBa_K3888005 SequenceAndFeatures</partinfo>