Difference between revisions of "Part:BBa K3888998"

Latest revision as of 17:28, 20 October 2021

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.

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.

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

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3888998 short</partinfo>
-Knockout of UppE in R. palustris
+UppE Knockout<br>
+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.  <br>
+[[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. <br>
+According to this model, we constructed UppE knockout plasmid below.
+[[File:UppE Deletion Plasmid Map.png]]
+===Figure 2: Deletion plasmid of UppE constructed according to the method mentioned above.===
 <!-- Add more about the biology of this part here
 ===Usage and Biology===