Difference between revisions of "Part:BBa K551001:Design"

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===Design Notes===
 
===Design Notes===
  
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We used yeast assembly to create pINDEL; we started from pKD46 that was assembled with pFL44S to make use of the yeast homologous recombination machinery (as shown with crosses in the first picture). Since this intermediate can only insert genes through the complete RED recombinase machinery we called it pIN.
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To construct pINDEL, we have proceeded in 2 steps and used the high capacity of yeast to perform homologous recombination.  The first step was to recombine a large IN fragment containing the repA101Ts thermosensitive replication origin, the exo, gam and bet genes under the control of the pBAD promoter as well as the araC gene encoding the AraC transcriptional regulator with another large fragment containing a yeast replication origin, the yeast selection marker ura3 and the Amp resistance gene. This first construct was named pIN since it is only able to insert genes.
 +
 
 
[[Image:pindel-yeast-1.png|center|700px|alt=yeast assembly of pKD46 with pFL44S to obtain pIN]]
 
[[Image:pindel-yeast-1.png|center|700px|alt=yeast assembly of pKD46 with pFL44S to obtain pIN]]
  
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Afterwards we assembled pIN and pPC20 to add the deletion possibilities of the flipase recombinase (as shown in the second picture).
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In a second step, we recombined the DEL unit composed of the flp gene under the control of the lambda pR promoter and of the cI857 gene encoding the thermosensitive CI repressor of pR to the pIN plasmid in yeast to finally obtain the pINDEL plasmid
 +
 
 
[[Image:pindel-yeast-2.png|center|700px|alt=yeast assembly of pIN with pPC20 to obtain pINDEL]]
 
[[Image:pindel-yeast-2.png|center|700px|alt=yeast assembly of pIN with pPC20 to obtain pINDEL]]
  

Revision as of 16:02, 21 September 2011

plasmid of insertion and deletion


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


Design Notes

To construct pINDEL, we have proceeded in 2 steps and used the high capacity of yeast to perform homologous recombination. The first step was to recombine a large IN fragment containing the repA101Ts thermosensitive replication origin, the exo, gam and bet genes under the control of the pBAD promoter as well as the araC gene encoding the AraC transcriptional regulator with another large fragment containing a yeast replication origin, the yeast selection marker ura3 and the Amp resistance gene. This first construct was named pIN since it is only able to insert genes.

yeast assembly of pKD46 with pFL44S to obtain pIN

In a second step, we recombined the DEL unit composed of the flp gene under the control of the lambda pR promoter and of the cI857 gene encoding the thermosensitive CI repressor of pR to the pIN plasmid in yeast to finally obtain the pINDEL plasmid

yeast assembly of pIN with pPC20 to obtain pINDEL

The homologous bits were obtained through PCR on the plasmid.

Source

The basic scaffold is pKD46 on which were added a yeast replication origin and yeast selection marker from pFL44S and the flipase of pPC20

References

"One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products" Datsenko KA, Wanner BL. PNAS 2000.