Experimental protocols for in-vivo cloning with mini-Mu in Salmonella
P22 strain construction with galE strains:
To generate a P22 lysate from a galE strain, or transduce a P22 lysate into a galE strain, the rest of the gal operon must be induced in order for P22 to attach and enter the cell. This is most easily accomplished by growing the galE strain in rich media supplemented with both .2% galactose, and .2% glucose. Although it is possible to grow galE strains directly in .2% galactose, .2% glucose, better results will be obtained by first starting a rich unsupplemented overnight of the galE strain, and then subculturing this overnight 1:400 in rich media supplemented with .2% galactose, .2% glucose. This subculture will take ~ 6 hrs to grow to saturation.
Note- Do not supplement rich media with only .2% galactose, and no glucose, as some of the gal precursors are toxic to the cell in the absence of the GalE protein. The presence of both galactose and glucose allows for the expression of the gal genes at an intermediate, non-toxic level.
TT10313 hut+ galE542 MuHP1 (Mu cts62 hP1-1)
This strain contains the hybrid Mu-P1 temperature sensitive lysogen necessary for generating Mu lysates.
TT17198 (TT10313 pBC4042)
This strain harbors a plasmid containing the mini-Mu cloning element MudII4042. The plasmid is AmpR, CamR, and the mini-Mu element contains CamR and the p15A replication origin. The maximal insert size of this mini-Mu is 22.3 Kb, and lac operon fusions are possible.
TT17199 (TT10313 pEG5155)
This strain harbors a plasmid containing the mini-Mu cloning element MudI5155. The plasmid is AmpR, KanR, and the mini-Mu element contains KanR and the pMB1 (colE1) replication origin. The maximal insert size of this mini-Mu is 23.4 Kb, and lac operon fusions are possible.
TT18XXX (TT10313 pEG5005)
This strain harbors a plasmid containing the mini-Mu cloning element MudI5005. The plasmid is AmpR, KanR, and the mini-Mu element contains KanR and the pMB1 (colE1) replication origin. The maximal insert size of this mini-Mu is 31.1 Kb, however, lac operon fusions are not possible. If very large inserts are desired, then this is the mini-Mu element of choice. However, one should keep in mind that larger inserts are harder to subclone.
Generating a mini-Mu lysate:
1- Grow a 30oC rich overnight of a MuHP1, mini-Mu dilysogen (TT17198, TT17199, or TT18XXX).
Note- Do not add any of the plasmid encoding drugs to the overnight, as the plasmid will be destroyed when Mu is induced.
2- Inoculate .075 ml of the overnight culture into 15 ml of rich media.
3- Incubate at 30oC with moderate shaking until the cell density reaches ~60-80 Klett units.
4- Transfer the culture to 44-45oC for 30 min and continue to shake moderately.
5- Shift the culture to 37oC and continue to shake moderately. Lysis should occur at approximately 1 1/2 - 2 hr after the shift to 44-45oC. Monitor the cell growth by taking Klett readings every 30 min until the lysis levels off.
Note- Dilysogen strains that contain a mini-Mu element sometimes do not lyse (high titers of Mu will be generated in these strains without lysis). For this reason it is helpful to monitor a simultaneously induced culture of TT10313 as a control. When this culture's lysis and levels off, maximal yield of Mu will have occurred in the dilysogen as well.
6- Add 2 ml of chloroform and continue moderate shaking for an additional 15 min.
7- Pellet the debris and chloroform. A clinical centrifuge is adequate for this spin.
8- Transfer the supernatant to a new tube, and do a 10 min, 8000 K spin at 5oC.
9- Transfer the supernatant to a new tube. This is the Mu transducing lysate.
Note- Mu lysates are stable for a week at best. It is absolutely imperative to determine the transduction forming capability of the lysate and use it as soon as possible. It is possible to stabilize Mu by adding EGTA to a final concentration of 5mM to the culture just after the downshift from 44-45oC to 37oC. This will result in a Mu lysate that is much more stable, however, the titer could be as much a 10-2 lower, if the EGTA is added.
Transducing with the Mu lysate:
1- Make a rich overnight, supplemented with .2% glucose, from a galE strain that contains Mu repressor. TT10313 is fine for this purpose, however galE strains that contain a MudA or MudJ insertion will work as well.
Note- Remember to grow TT10313 and MudA or MudJ containing strains at 30oC, as these all harbor Mucts. The recipient strain must contain functional Mu repressor so that the incoming mini-Mu element does not transpose.
2- Pellet the overnight and resuspend the cells in 1/2 volume of 10mM MgSO4, 5mM CaCl2.
3- Mix 100 ml of concentrated, 10-1, and 10-2 Mu lysate (use rich media for the dilution) with 100ml of resuspended cells. Also include 100ml of concentrated lysate, and 100ml of resuspended cells as controls.
4- Incubate at 30oC without shaking for 45 min.
5- Plate the samples on a rich plate which contains the drug that is encoded by the mini-Mu cloning element being used.
6- Incubate the plates at 30oC.
Note- It usually takes two days to obtain colonies.
Last Update: Thursday, 19-Jun-2014 12:34:08 PDT
This page has been viewed 108 times.
eckofoid at ucdavis.edu