Converting Muds and Making Mud-Mud Rearrangements
Mud held duplications or deletions are a useful tool in themselves, and the ability of form such rearrangements is used to orient Muds and the transcription unit they are in. Such rearrangements are constructed using MudAs because their large size requires two fragment transductions. Since Mud insertions are usually isolated as MudJ hops these days, the MudJs must first be converted to MudAs by replacing the guts of the transposon via homologous recombination within the transposon borders (see the list of Muds included here for the available homologies). Most old fusions in the collection are MudAs, and it is often convenient to convert these to MudJs if they are commonly used.
1) A donor is used which has the replacing Mud version in either an E. Coli F' so there is no homology outside the Mud for the drug to be inherited with , or with the Mud in an essential gene where the recombinants outside the Mud are easy to select against. Possible donors are:
TT purA::MudJ don't grow on NB
TT purA::MudA don't grow on NB
TT hemA::MudA don;t grow on NB
TT12000 hemA::MudJ don't grow on NB
2) The hemA fusions are nice since on a NB drug plate there are many very small colonies that represent the recombinents at hemA, these let you know the transduction is working at a reasonable frequency, and a few large colonies that are the Mud replacements.
3) After the transduction, clean up the colonies on green plates, and check to make sure the first drug is gone and that proper regulation has been retained.
Making Duplications and Deletions
This just requires doing a transduction with a mixed lysate, using phage from two donor MudAs that are in the same orientation. There is a list of MudAs around the chromosome included here. Select for AmpR and score for the phenotypes of the two parent Muds. Duplications are wt and segregrate the Mud, deletions (if possible) have both parental deficiencies and are stable. Both parental insertions will be regenerated as well; the ratio of DUP:DEL:Parental is about 1:1:2. Mud held duplications and deletions can be transduced by selecting for the Mud, this is the final test of a construction
Last Update: Thursday, 19-Jun-2014 12:13:02 PDT
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