Download M9 salts (1 liter)

BAC Recombineering in SW102
I. One-step deletions in BAC clones
1. Obtain and characterize a suitable BAC clone. Transform into SW102.
Transformation must be by electroporation, to avoid heat shocking. Recovery after electroporation is for 1 hour at 30°C. Plate on LB + 12.5 mg/ml chloramphenicol (CAM).
Incubate plates at 30°C.
2. Design kan primers with 50 bp homology to an area flanking the deletion site.
The 3’ ends of the primers are kan sequence (green):
Forward: 5’----50bp homology------- GAGTCGTATTACATGGTCATAGC-3’
Reverse: 5’----50bp homology (compl. strand)---- GTCCCGTCAAGTCAGCGTAAT-3’
3. PCR amplify the kan cassette
Use a proof-reading polymerase and 1-2 ng plasmid.
30 cycles of 94°C 15 sec., 60°C 30 sec, 72°C 1 min
Remove plasmid template: digest with 1-2 μl DpnI per 25 μl, 37°C for 1 hour.
Gel-purify the DpnI-digested PCR product. Remove buffer by precipitation or column
purification, and resuspend in water.
4. Electroporate induced SW102/BAC cells with the kan cassette.
See protocol on last page. Use 10-30 ng purified PCR product per transformation.
5. Plate on LB + Kan plates.
Incubate plates at 30°C. Check colonies by colony PCR. To make BAC DNA for
transformation, first recover DNA from SW102 by mini-prep, then transform into EPI300 for
copy control amplification.
BAC Recombineering in SW102
II. Copying sequences from a BAC to another vector
1. Obtain and characterize a suitable BAC clone. Transform into SW102.
Transformation must be by electroporation, to avoid heat shocking. Recovery after electroporation is for 1 hour at 30°C. Plate on LB + 12.5 mg/ml chloramphenicol (CAM).
Incubate plates at 30°C.
2. Clone homology arms into the recipient vector.
The recipient vector must have an antibiotic selection other than CAM. You will need to
clone homology arms into this vector to support gap repair during recombineering. The
diagram below shows the basic design. Each homology arm is 500 bp (or more) at the ends
of the region to be copied, cloned into the recipient vector (gray). R = restriction
endonuclease site, unique within the clone. Digest with this enzyme and gel purify the
linearized vector before transformation.
R
left arm
right arm
3. Electroporate induced SW102/BAC cells with the kan cassette.
See protocol on last page. Use 10-30 ng purified vector per transformation.
4. Plate on LB + antibiotic (selective for the recipient vector) plates.
Incubate plates at 30°C. Check colonies by colony PCR. Recover DNA and transform into
another host strain for large-scale preps.
III. Using galK positive/negative selection
The galK positive and negative selection scheme can be used to make point mutations,
deletions, and insertions. SW102 contains the  prophage recombineering system; the
galactokinase gene (galK) has been deleted. The galK selection scheme is a two-step system:
First, a cassette containing galK flanked by arms that are homologous to the target site in the
BAC is inserted into the BAC. Recombinant bacteria are selected by being able to grow on
minimal medium with galactose as the only carbon source. Second, the galK cassette is
substituted by an oligo, a PCR product, or a cloned fragment with homology flanking the
cassette. Recombinants are identified by selecting against galK by resistance to 2-deoxygalactose (DOG) on minimal plates with glycerol as the carbon source. DOG is harmless
unless phosphorylated by GalK, which turns it into a non-metabolizable, toxic intermediate.
A. DESIGN THE PROJECT
1. Obtain and characterize a suitable BAC clone.
2. Design galK primers with 50 bp homology to an area flanking the modification site.
Point mutation: Homology arms should be the 50 bp on either side of the single bp.
This will result in a deletion of that base pair in the first step.
Deletion: Design the galK primers so the deletion is made already in the first step.
Insertion: Design the primers so the 50 bp arms abut the insertion site.
The 3’ ends of are galK sequence (blue):
Forward: 5’----50bp homology-------CCTGTTGACAATTAATCATCGGCA-3’
Reverse: 5’----50bp homology (compl._strand)----TCAGCACTGTCCTGCTCCTT-3’
3. Design replacement DNA.
Point mutation: Make two 100 nt complementary oligos with the modified basepair(s) in
the middle; the remaining bases on either side are homology arms.
Deletion: Make two 100 nt complementary oligos that span the deleted sequence (i.e.,
50 nt on each side), to produce a “seam-less” deletion.
Insertion: For small insertions, such as an epitope tag, you can design complementary
oligos that have 50 nt flanking homology plus the tag in the middle (note that these are
>100 nt each). For larger insertions, you can PCR amplify the insertion sequence with
flanking homology arms, exactly as done for galK in step 2. Another method is to clone
the insertion into the correct site in another vector, then use either that vector or a PCRamplifed fragment as the donor in the second stage.
B. INSERT GALK, USING POSITIVE SELECTION.
1. PCR amplify the galK cassette
Use a proof-reading polymerase and 1-2 ng pgalK plasmid.
30 cycles of 94°C 15 sec., 60°C 30 sec, 72°C 1 min
To remove plasmid template, digest with 1-2 μl DpnI per 25 μl, 37°C for 1 hour.
Gel-purify the DpnI-digested PCR product. Remove buffer by precipitation or column
purification, and resuspend in water. Use 10-30 ng for a transformation.
2. Transform the BAC to be modified into electrocompetent SW102 cells
Recover for 1 hour at 32°C, and plate on LB + 12.5 mg/ml chloramphenicol.
Grow plates at 32°C.
3. Electroporate induced SW102/BAC cells with the galK cassette.
See protocol on last page.
4. Plate on selective minimal medium.
a. Wash the transformed bacteria twice in M9 salts:
Pellet the 1 ml culture in an Eppendorf tube at full speed for 15 sec.
Remove supernatant removed with a pipette and resuspend in 1 ml M9 salts.
Pellet again , remove supernatant, resuspend in 1 ml M9 salts.
b. Plate serial dilutions onto M63 plates with galactose, leucine, biotin, and CAM.
100 μl , 100 μl of a 1:10 dilution in M9, and 100 μl 1:100 dilution in M9.
The uninduced samples routinely have a higher degree of lysis/bacterial death
after electroporation, so this sample is resuspended in 0.25 – 0.75 ml M9 salts in
the final step and plate 100 μl.
c. Incubate for 3 days at 32°C.
7. Streak a few colonies onto MacConkey + galactose + CAM.
Incubate overnight at 32°C. This step removes Gal- contaminants (hitch-hikers):
Gal- colonies will be white/colorless and the Gal+ bacteria will be bright red/pink.
C. REPLACE GALK, USING NEGATIVE SELECTION.
1. Prepare the replacement DNA.
Either PCR amplify as in step 1 above, or anneal oligos (final concentration of 200 ng/ μl,
use 1 μl per transformation).
2. Grow an overnight culture of the galK-insertion BAC.
Pick a single, bright red (Gal+) colony and inoculate a 5 ml LB + CAM. Incubate at
32°C. There is normally no need to further characterize the clones after the first step,
though it would be easy to do a colony PCR to verify the correct insertion of galK.
3. Electroporate induced BAC/galK cells with the replacement DNA.
Repeat step 3 above, but recover at 32°C for 4.5 hours.
4. Plate on selective minimal medium.
Repeat step 4 above, but plate on M63 with glycerol, leucine, biotin, 2-deoxy-Dgalacytose, and CAM. Analyze 10-12 colonies. Background clones (DOG resistant
without the desired mutation) arise by spontaneous deletion of the galK gene and flanking
DNA, which should be obvious. If the background is too high, try increasing the length of
the homology arms (33 bp gives an efficiency of 50% correct; 50 bp gives 80%).
C. REPLACE GALK, USING NEGATIVE SELECTION.
Premixed media can also be purchased.
M63 minimal plates
1L 5X M63
10 g (NH4)2SO4
68 g KH2PO4
2.5 mg FeSO4·7H2O
adjust to pH 7 with KOH
AUTOCLAVE
M9 salts (1 liter)
6 g Na2HPO4
3 g KH2PO4
1 g NH4Cl
0.5 g NaCl
AUTOCLAVE
0.2 mg/ml d-biotin (sterile filtered) (1:5000)
20% galactose (autoclaved) (1:100)
20% 2-deoxy-galactose (autoclaved) (1:100)
20% glycerol (autoclaved) (1:100)
10 mg/ml L-leucine (1%, heated, cooled, filtered)
25 mg/ml Chloramphenicol in EtOH (1:2000)
1 M MgSO4·7H2O (1:1000)
Autoclave 15 g agar in 800 ml H2O in a 2 liter flask. Let cool down a little.
Add 200 ml autoclaved 5X M63 medium and 1 ml 1 M MgSO4·7H2O.
Adjust volume to 1 liter with H2O if necessary. Let cool down to 50°C (“touchable hot”).
Add 10 ml carbon source (final conc. 0.2%)
5 ml biotin (1 mg)
4.5 ml leucine (45 mg)
500 l Chloramphenicol (final conc. 12.5 g/ml).
MacConkey indicator plates
Prepare MacConkey agar plus galactose according to manufacturer’s instructions. After
autoclaving and cooling to 50°C, to one liter add 500 ml chloramphenicol (CAM; final conc. 12.5
mg/ml).
Electroporation [and Induction] of SW102
This is a combination of the basic electroporation protocol (washing bacteria into ddH2O) and
the heat-shock protocol used to induce λ red recombination for recombineering. For
recombineering steps, include the steps in brackets (heat shocking at 42°C). To just transform
a BAC into SW102, skip the heat-shock procedure.
1. Inoculate an overnight culture of SW102 cells containing the BAC into 5 ml LB + CAM (12.5
μg/ml). Incubate at 30°C overnight.
2. Dilute 500 μl of the overnight culture in 25 ml LB with CAM. Incubate at 30°C, shaking, to
an OD600 of 0.6 (± 0.05). This usually takes 3-4 hours at 30°C.
3. During this incubation, put a 50 ml tube of ddH2O into a slurry of ice and water.
[If inducing recombination, turn on a water bath for 42°C.]
[4. For induction of recombination, transfer 10 ml of the culture to a 50 ml conical tube and
heat-shock at 42°C for 15 min. This is supposed to be in a shaking water bath, so shake it
occasionally. The remaining culture is left at 30°C as the un-induced control (if you do that
sort of thing).]
5. Briefly cool the culture(s) in an ice/water slurry, then transfer 10 ml to 15 ml Falcon tubes
and pellet at 3000 RPM at 4°C for 5 min. The Matson tabletop centrifuge in cold room is
perfect. It’s important to keep the bacteria as close to 0°C as possible in order to get good
competent cells.
6. Pour off the supernatant and resuspend the pellet in 1 ml ice-cold ddH2O by gently swirling
the tubes in the ice/water slurry (do not pipette). This may take some time.
7. Add another 9 ml ice-cold ddH2O, pellet again. Pour off the supernatant and allow it to drain
off briefly. Resuspend in whatever liquid is left, typically about 50 μl.
8. Electroporate 40 of μl cells, standard settings. Recover for 1 hr at 30°C before plating.
Incubate plates at 30°C.