Difference between revisions of "Part:BBa K422015"
(→‎Characterization)
 
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Victor de Lorenzo's lab, Ampicillin resistance, pBBR1 ori  
 
Victor de Lorenzo's lab, Ampicillin resistance, pBBR1 ori  
 +
  
 
===Plasmid features===
 
===Plasmid features===
 +
* Ampicillin resistance
 +
* pBBR1 origin of replication
 +
 +
 +
===Characterization===
 +
[[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 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===
 
===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.
  
  

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