Difference between revisions of "Part:BBa K551001"
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We performed a thermal sensitivity experiment in liquid medium in which we measured the plasmid loss frequency as a function of time of culture at 42°C. We compared the viability (CFU/ml: colony forming units per ml of culture) of MC1061/pIN grown at 30°C and 42°C and plated on LB and LB Amp medium. If the plasmid is lost, then the bacteria won't grow on the LB Amp medium plates. | We performed a thermal sensitivity experiment in liquid medium in which we measured the plasmid loss frequency as a function of time of culture at 42°C. We compared the viability (CFU/ml: colony forming units per ml of culture) of MC1061/pIN grown at 30°C and 42°C and plated on LB and LB Amp medium. If the plasmid is lost, then the bacteria won't grow on the LB Amp medium plates. | ||
Revision as of 16:21, 21 September 2011
plasmid of insertion and deletion
The pINDEL plasmid is composed of 2 functional units:
(i) the IN function which is composed of the gam, exo and bet genes coding for the lambda Red recombinase system (ii) the DEL function which is based on the flp gene encoding the FLP site-specific recombinase and expressed at 42°C
Another feature of pINDEL is that its replication origin is not functional at 42°C due to the thermosensitivity of the RepA101Ts protein. This protein initiates replication at the ori site in permissive conditions (30°C) and is inactive at 42°C.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 396
Illegal EcoRI site found at 3708
Illegal EcoRI site found at 5217
Illegal EcoRI site found at 6322
Illegal XbaI site found at 423
Illegal SpeI site found at 6004
Illegal SpeI site found at 8956
Illegal PstI site found at 435
Illegal PstI site found at 4712
Illegal PstI site found at 4959 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 396
Illegal EcoRI site found at 3708
Illegal EcoRI site found at 5217
Illegal EcoRI site found at 6322
Illegal SpeI site found at 6004
Illegal SpeI site found at 8956
Illegal PstI site found at 435
Illegal PstI site found at 4712
Illegal PstI site found at 4959
Illegal NotI site found at 9275 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 396
Illegal EcoRI site found at 3708
Illegal EcoRI site found at 5217
Illegal EcoRI site found at 6322
Illegal BglII site found at 632
Illegal BglII site found at 1728
Illegal BamHI site found at 417
Illegal BamHI site found at 3647 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 396
Illegal EcoRI site found at 3708
Illegal EcoRI site found at 5217
Illegal EcoRI site found at 6322
Illegal XbaI site found at 423
Illegal SpeI site found at 6004
Illegal SpeI site found at 8956
Illegal PstI site found at 435
Illegal PstI site found at 4712
Illegal PstI site found at 4959 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 396
Illegal EcoRI site found at 3708
Illegal EcoRI site found at 5217
Illegal EcoRI site found at 6322
Illegal XbaI site found at 423
Illegal SpeI site found at 6004
Illegal SpeI site found at 8956
Illegal PstI site found at 435
Illegal PstI site found at 4712
Illegal PstI site found at 4959
Illegal AgeI site found at 3482
Illegal AgeI site found at 5139 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 837
Illegal BsaI.rc site found at 9958
Illegal SapI site found at 989
Illegal SapI site found at 1787
Illegal SapI site found at 3464
Functional Parameters
We performed a thermal sensitivity experiment in liquid medium in which we measured the plasmid loss frequency as a function of time of culture at 42°C. We compared the viability (CFU/ml: colony forming units per ml of culture) of MC1061/pIN grown at 30°C and 42°C and plated on LB and LB Amp medium. If the plasmid is lost, then the bacteria won't grow on the LB Amp medium plates.