Part:BBa_K551001

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

Assembly Compatibility:

• 10
  INCOMPATIBLE 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
  
• 12
  INCOMPATIBLE 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
  
• 21
  INCOMPATIBLE 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
  
• 23
  INCOMPATIBLE 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
  
• 25
  INCOMPATIBLE 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
  
• 1000
  INCOMPATIBLE 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

resistance

100 �g/ml

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.