Plasmid
pINDEL

Part:BBa_K551001

Designed by: ULB-Brussels team   Group: iGEM11_ULB-Brussels   (2011-09-19)
Revision as of 16:31, 21 September 2011 by ULB (Talk | contribs) (→‎Functional Parameters)

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.

The deletion function and the thermosensitivity of the plasmid both could be shown to be working as intended. The insertion function couldn't be properly tested.

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

Thermosensitivity

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
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.

Deletion function

To test the deletion capacity of the pINDEL plasmid, we transformed pINDEL and pIN in the MG1655 ∆tldD ::frt-cm-frt strain and the transformants were selected on LB Amp Cm medium at 30°C. Four independant colonies containing pIN or pINDEL were streaked on LB plates and were incubated ON at 42°C to lose plasmids and to express the flipase. For each independant transformants streaked we stabbed 8 colonies on LB and LB Cm plates.

All 32 candidates that contained pIN grew on LB plates and LB Cm plates indicating that none of them has lost the antibiotic resistance cassette. All 32 candidates that contained pINDEL grew on LB but none of them grew on LB Cm plates indicating that all of them have lost the antibiotic resistance cassette.

This gives us a 100% success rate for excision.

Growth

[edit]
Categories
//function/recombination/flp
Parameters
resistance100 g/ml