Difference between revisions of "Part:BBa K422015"

Latest revision as of 10:36, 27 October 2010

pSEVA132

Victor de Lorenzo's lab, Ampicillin resistance, pBBR1 ori

Plasmid features

Ampicillin resistance
pBBR1 origin of replication

Characterization

Figure 1: Plasmid copy number estimation of pSEVA132 compared to the high copy plasmid pUC19.

As pBBR1 is not a widely known origin of replication, the plasmid copy number was determined in relation to a commonly used high-copy vector pUC19. The experimental procedure included normalization of cell number via optical density measurement followed by plasmid concentration measurements (using a commercial Miniprep kit). The results are represented in figure 1.

Design features

Compatible with the cloning strategy BBF RFC28.

The genes for the repressor protein AraC and the corresponding ParaBAD promotor/operator sites were introduced into the vector pSEVA132 followed by an insert, which is flanked by AarI-recognition sites. Digest with AarI releases this insert and generates a vector with assembly-compatible overhangs.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 292
Illegal EcoRI site found at 1505
Illegal XbaI site found at 3478
Illegal SpeI site found at 1
Illegal SpeI site found at 3127
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 292
Illegal EcoRI site found at 1505
Illegal SpeI site found at 1
Illegal SpeI site found at 3127
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 292
Illegal EcoRI site found at 1505
Illegal BglII site found at 1899
Illegal BamHI site found at 1464
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 292
Illegal EcoRI site found at 1505
Illegal XbaI site found at 3478
Illegal SpeI site found at 1
Illegal SpeI site found at 3127
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 292
Illegal EcoRI site found at 1505
Illegal XbaI site found at 3478
Illegal SpeI site found at 1
Illegal SpeI site found at 3127
Illegal NgoMIV site found at 5397
Illegal NgoMIV site found at 5891
Illegal NgoMIV site found at 6020
Illegal AgeI site found at 1299
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 1597
Illegal SapI site found at 1281
Illegal SapI site found at 3733
Illegal SapI.rc site found at 3061
Illegal SapI.rc site found at 3409

@@ Line 11: / Line 11: @@
 ===Characterization===
-[[Image:Copy number.png|thumb|200px|'''Figure 1:''' Plasmid copy number estimation.]]
+[[Image:Plasmid_copy_number.png|thumb|200px|'''Figure 1:''' Plasmid copy number estimation of pSEVA132 compared to the high copy plasmid pUC19.]]
-As pBBR1 is not a widely known origin of replication, the plasmid copy number was determined by the normalization of cell number via optical density measurement followed by plasmid concentration measurements (using a commercial Miniprep kit). The results are represented in figure 1.
+As pBBR1 is not a widely known origin of replication, the plasmid copy number was determined in relation to a commonly used high-copy vector pUC19. The experimental procedure included normalization of cell number via optical density measurement followed by plasmid concentration measurements (using a commercial Miniprep kit). The results are represented in figure 1.
 ===Design features===
 Compatible with the cloning strategy BBF RFC28.
-The genes for the repressor protein AraC and the corresponding ParaBAD promotor/operator sites were introduced into the vector pSEVA 132 followed by an insert, which is flanked by AarI-recognition sites. Digest with AarI releases this insert and generates a vector with assembly-compatible overhangs.
+The genes for the repressor protein AraC and the corresponding ParaBAD promotor/operator sites were introduced into the vector pSEVA132 followed by an insert, which is flanked by AarI-recognition sites. Digest with AarI releases this insert and generates a vector with assembly-compatible overhangs.