Deletion procedures step by step
Procedure 1. (using pBAD and pKSUC1)
I. Construction of a linear targeting fragment by PCR.
1. Mix 20 pmol primer a with 20 pmol primer b and perform PCR in a total volume of 50 l.
Cycle parameters: 15x(94oC 40sec/57oC or lower [depending on the overlap of a and b]
40sec/72oC 15sec).
2. Take 1 l of the PCR mix above, add 20 pmol of primers a and c each, add 50 ng of pSG76CS and perform PCR in a volume of 2x50 l. Cycle parameters: 28x(94oC 40sec/57oC
40sec/72oC 80sec).
3. Purify the PCR mix from I/2 with Promega Wizard PCR purification kit. Suspend the
fragment in 20 l water.
II. Replacement of a genomic region by insertion of the DNA-fragment.
1. Take the target cell carrying pBAD and prepare electrocompetent cells as described
(Pósfai et al., Nucleic Acids Res, 1999, 27, 4409-4415) except that 0.1% L-arabinose is
added to the culture 0.25 -1 hour before harvesting the cells.
2. Electroporate 4 l DNA-fragment (100-200 ng) from I/3 into 40 l of electrocompetent cells.
Plate the cells on Cm plates (25 g /ml) and incubate at 37oC. We usually obtain a total of 10
to several hundred colonies after overnight incubation.
3. Check a few colonies for correct site insertion of the fragment by PCR using primers d and e.
III. Deletion of the inserted sequences.
1. Prepare competent cells derived from a selected colony from II/3.
2. Transform pKSUC1 (~100 ng) into the cells by standard procedures. Plate the cells on Km
plates and incubate at 37oC. (pKSUC1 and pBADare incompatible, thus, selection on
Km eliminates pBADfrom the cells.)
3. Check colonies for correct deletion by PCR using primers d and e. Select a colony carrying
the correct deletion. At this point, the cell carries pKSUC1. To get rid of the plasmid, go to
step IV.
IV. Replacement of pKSUC1 with pBAD.
1. Take a selected colony from III/3, grow it in LB at 37oC under nonselective conditions
(without Km) , preferably reinoculating cells into fresh medium 2-3 times. Prepare competent
cells for either chemical transformation or electroporation. Transform pBAD (100-200
ng) into the competent cell and plate it on Ap plates. Select a colony which is Km
sensitive/Ap resistant by toothpicking a hundred colonies on Km and Ap plates.
2. The selected colony can be used in a next round of deletion by using a new targeting
fragment and repeating steps from II/1. If no more deletions are needed, growing the cells
under nonselective conditions (no Ap is added) results in the spontaneous loss of
pBADfrom a large fraction of the cells.
Notes:
1. Efficiency of the insertion of the linear fragment varied with the particular genomic locus.
Correct-site insertion occurred in 5-100% of the colonies. Flanking homologies in the range
of 40 to 80 bp were used. Longer homologies give better insertion efficiencies.
2. In step III, deletion of the inserted sequence occurs in nearly every cell. However, only a
fraction of the cells shows the predicted recombination between the duplicated regions. The
rest of the cells probably loose the inserted sequence via recombination between randomly
occurring, partially homologous regions flanking the insertion. Correct-site excision
between the duplicated sequences occurred in 10-100% of the colonies and depended on
the length of the duplicated region. Longer duplications are usually more effective. Length
of the duplicated sequences was in the range 40 to 60 bp. Variations in the efficiencies of
insertion and excision existed between seemingly identically repeated experiments for
reasons not fully understood yet.
3. L-arabinose stock: 10% in water, sterilized by filtration.
Procedure 2. (using pBADand pSTKST)
I. Construction of a linear targeting fragment by PCR.
Identical to step I. in Procedure 1.
II. Replacement of a genomic region by insertion of the DNA-fragment.
Identical to step II. in Procedure 1.
III. Transformation of pSTKST.
1. Prepare competent cells derived from a selected colony from II/3.
2. Transform pSTKST (100 ng) into the cell by standard procedures. Plate the cells on
Km+Cm plates and incubate at 30oCIV. Deletion of the inserted sequences.
1. Take a colony from III/2 and inoculate into10 ml of LB+Km supplemented with heattreated inducer cTc (25 g/ml final concentration) and grow at 30oC for 24 hours.
2. Plate dilutions of the culture (10-5-10-6-fold) on LB+Km plates and incubate overnight at
30oC.
3. Check 6-12 colonies for correct deletion by PCR using primers d and e. Select a colony
carrying the correct deletion.
V. Helper plasmids can be eliminated from the cells by growing the culture at 37-43oC in LB.
Replication of pSTKST is inhibited at this temperature and pBAD is rapidly lost from
the cells under non-selective conditions (no Ap added).
Notes:
1. L-arabinose stock: 10% in water, sterilized by filtration.
2. cTc stock: Suspended at 500 g/ml concentration in LB. Heat-treated in a pressure cooker
for 20 min and stored at 4oC in the dark.