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

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.

The MC1061 strain containing the pIN plasmid was grown in LB medium at 30°C (blue triangle) and 42°C (red square). At times indicated in the figure, a sample of the cultures was diluted and plated on LB and LB Amp plates as described in Materials and Methods. Colonies were counted after ON incubation at 30°C. The percentage of bacteria containing pIN is the ratio between the CFU/ml on LB Amp plates and the CFU/ml on LB plates multiplied by 100

This shows that the replication of pIN is impaired at 42°C. After 150 min of culture at 42°C, 60% of the bacteria have lost the pIN plasmid. After 275 min, only a very small proportion of bacteria retained the pIN plasmid (< 10%). Note that at time 0 of the experiment, around 70% of the bacteria retained the pIN plasmid suggesting that the repA101ts origin is not fully active even at 30°C. With time, the pIN plasmid is maintained in about 80% of the bacteria at 30°C. This experiment was performed only once due to time limitations and should be repeated to confirm the data.