LABMANUAL

Site specific insertion of GFP into BAC clones

Cathy Yuh, Sagar Damle

This method is derived from Yu et al. (2000). It employs a bacterial strain (EL250) derived from DH10B that carries a bacteriophage lambda prophage with the genes exo, bet and gam under the control of a temperature-sensitive c cI-repressor. These genes are off at 32°C and on at 42°C. Gam inhibits RecBCD nuclease from degrading linear DNA, and Exo and Beta provide double stranded break repair recombinase activity. The strain also carries a copy of the S. cerevisiae Flp recombinase that is inducible with arabinose. A plasmid containing a recombination cassette (pGFK) contains GFP-frt-Kan-frt surrounded by cloning sites. I have been cloning  100 bp PCR fragments from the BAC of interest around the ”GFP-frt-Kan-frt” cassette, then amplifying the region using the Roche Expand High Fidelity PCR System (cat-no: 1 732 650) from plasmid minipreps. Presumably 45 bp fragments will give similar recombination efficiencies so oligonucleotide-based approaches should work well. These can either be cloned or used to construct the cassette purely with PCR, although in the latter case it is difficult to verify the sequence of the cassette prior to recombination.

Making GFP-Km cassette with homologous region on each side

Cloning method:

Design PCR primers that match to the 5’ and 3’ ends of the first exon. You will need two sets of primer-pairs. Each pair will amplify either the 5’ (leading) or 3’ (trailing) end of the first exon. Of the leading-end primer-pairs, the 5’ primer should be tailed with a SacI (GAGCTC) restriction site and the 3’ primer should be tailed with a SpeI (ACTAGT) site. Of the trailing-end primer-pairs, the 5’ and 3’ primers should be tailed with SalI (GTCGAC) and KpnI (GGTACC) respectively. You also need to order a pair of PCR primers that have no restriction sites attached to them and that amplify the target site. In other words, order the same sequence as the SacI-tailed and Kpn1-tailed primers - but this time do not include the SacI or KpnI restriction sites in the sequence. This primer pair will be used to amplify the cassette for recombination. Finally, you will need another set of primers that are flanking and outside of the recombination site. Use these to check that the cassette has been correctly inserted into the target sequence (See note 1 for example of SpOtx).

PCR method:

Design primers that amplify the cassette and that have at least a 45bp tail that matches the target site for homologous recombination. If the goal is to replace the first exon of a coding sequence with cassette sequence, then design primers whose tails match the 5’ and 3’ ends of that exon. In this example, design a 45 bp tailing GFP primer (5’) and 45 bp tailing Kan primer (3’). Using a GFP-F(forward) primer and Kan-R(reverse) primer, we generate a 2737bp cassette. But with this protocol, we can use any cassette plasmid DNA as template because it is the PCR amplification that generates the target-specific cassette. (see note 2 for example of LvOtx).

PCR condition is very important in this method. You need to have a final MgCl2 concentration of 2mM and 60°C annealing temperature.

Per 50ml of PCR reaction (Roche Expand High Fidelity PCR System, cat-no: 1 732 650):

5ml of (10ng/ml) cassette plasmid
2ml of 10mM primer1
2ml of 10mM primer2
5ml of 10X buffer (2)
1ml of 10mM dNTP
2ml of 25mM MgCl2 (4)
0.75ml of enzyme (1)

Use the following program:

94°C 4min



94°C 1min
60°C 1 min 30 cycles
72°C + 5s/cycle



72°C 10min
C soak

After PCR, cleanup with Quiagen PCR mini-elute column, and digested with DpnI for 1 hour at 37°C, and cleanup again.

Making Electrocompetent EL250 cells

  1. Grow a 2 ml culture of EL250 cells overnight in LB at 32°C.
  2. Inoculate a fresh 50ml LB culture with 1ml of the 2ml culture and continue to grow at 32°C to O.D. 600 = 0.4-0.6 (4-5hr). Use a sterile 250ml flask.
  3. Spin down in 50ml Falcon tube in J6 centrifuge at 3000rpm 15min.
  4. Resuspend in 50ml ice-cold dH2O and spin down.
  5. Resuspend in 5 ml ice-cold dH2O and transfer to eppendorf tube. 1ml into each eppendorf tube, so you will have five tubes from 50 ml culture.
  6. Wash 3 more times in dH2O in 4°C microcetrifuge. Use 1ml of ice-cold sterile water to resuspend the cell pellet, and spin for 20s at 4°C at maximum speed in a microfuge.
  7. Use 100ml of water to resuspend the pellet from each tube, and use 50ml for electroporation, which is about 108 cells per reaction. Glycerin can be added to 12% and cells stored at -80°C. Fresh competent cells give highest efficiencies.

Electroporating BAC DNA into EL250 cells

  1. On ice, add 1ml DNA from a fresh BAC miniprep (300-600 ng) to 50ml electrocompetent EL250 cells. Place in a cold 0.1 mm cuvette. Electroporate at 1.4kV and immediately place in 1 ml room temperature LB.
  2. Grow at 32°C for 1.5h.
  3. Plate 50ml and 500ml on LB cam (chloramphenicol) plates and grow overnight at 32°C. Growth is slow at this temperature so 24h may be necessary. To be certain that you get a transformed colony, culture 500ml of transformed EL250 cells in 2.5ml LB cam overnight at 32°C. Plate 5ml onto an LB cam plate.
  4. Grow a few colonies, PCR check to see if the electroporated BAC contains the target site for recombination. Miniprep, and check by Not1 digestion to be sure that the BAC is intact.
  5. Make a glycerol stock (225ml glycerol + 1275ml miniprep culture) and store at -80°C.

Recombining cassette with BAC DNA

  1. Grow an overnight miniprep of EL250-BAC cells at 32°C in LB cam.
  2. Inoculate 50ml LB cam with 1ml fresh overnight culture. Grow at 32°C to O.D. 600 = 0.4 - 0.6 (4 to 5 hours).
  3. Place in pre-warmed 42°C water bath with shaking (200rpm) for exactly 15min to induce c recombinase activity.
  4. Immediately chill in ice-water slurry to 4°C (10 min).
  5. Place in a 50ml Falcon tube and spin down at 5,500g (3000rpm) for 15min at 4°C.
  6. Resuspend in 50ml ice-cold dH2O and spin down.
  7. Resuspend in 5ml ice-cold dH2O and transfer to eppendorf tube. 1ml into each eppendorf tube, so you will have five tubes from 50ml culture.
  8. Wash 3 more times in dH2O in 4°C microcentrifuge. Use 1ml of ice-cold sterile water to resuspend the cell pellet, and spin for 20s, 4°C at maximum speed in a microfuge.
  9. Use 100ml of water to resuspend the pellet from each tube, and use 50ml for electroporation, which is about 108 cells per reaction. Glycerin can be added to 12% and cells stored at -80°C. Fresh competent cells give highest efficiencies.
  10. On ice, add 1ml cassette DNA (300ng/ml) (note: if transformation with the template DNA will generate the selected phenotype (i.e., the template is a plasmid), the template must be eliminated. Plasmid template DNA can be destroyed by treatment with DpnI after PCR; DpnI cuts methylated GATC template DNA, leaving the newly replicated unmethylated DNA intact. Never use enzyme digested cassette DNA as the sourse for recombination, because the bacteria have the capacity to phosphorylate and ligate back the fragment.
  11. Place 50ml electrocompetent/recombination competent EL250-BAC cells in a cold 0.1mm cuvette. Electroporate at 1.70 kV and immediately place in 1ml room temperature LB.
  12. Grow at 32°C for 1h to express Kanamycin (kan) resistance.
  13. Plate 50ml and 500ml on LB cam/kan plates and grow overnight at 32°C. Growth is slow at this temperature so 24h may be necessary.
  14. Grow about 10 colonies, strip them on an Ampicillin plate to screen out those colonies that contain plasmid. After screening, choose those that can’t grow on the Ampicillin plate, using colony PCR to check if the bacteria contain the recombinant sequence (GFP-Km cassette). Using outside primer pairs check that the recombination happens at the right site. Miniprep, and cut with Not1 to be sure that BAC is intact and recombination has accurately taken place. The Not1 site in the cassette (just before the GFP start of translation) will allow cleavage of the insert at the site of integration. Integration can be further checked by PCR from the BAC sequence into the cassette and by sequencing out from the cassette. If you used the DpnI digest of the plasmid DNA or used very little template DNA as template for PCR, and also performed a gel-elution, more than 90% of colonies should appear to be correctly recombined as assayed by restriction digest.
  15. Make glycerol stock (225ml glycerol + 1275ml miniprep culture) and store at -80°C.

Activation of Flip recombinase to remove kan cassette

  1. Start a 25 ml LB cam culture using a 1:50 dilution (0.5ml) of a relatively fresh (<2 day) cam/kan culture from the primary recombinant in EL250 cells. Grow at 32°C.
  2. When the culture reaches O.D.600 = 0.5, add 0.25 ml 10% Arabinose for a final concentration of 0.1%. The arabinose solution should be made in dH2O, filtered and stored at 4°C. Grow for 1h.
  3. Dilute 1:10 (1ml in 9ml of LB cam in a 50ml Falcon tube) and grow one more hour.
  4. Steak out 2ml of this culture on LB cam plates. Grow at 32°C.
  5. Test for Kan sensitivity by growing individual colonies in minipreps of both LB cam and LB cam/kan. Typically >90% of colonies have lost Kan sensitivity. Clones that pass this test can be further analyzed by PCR and sequencing. A single frt site should remain in place of the frt-kan-frt region.

Reference:

Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, Court DL. 2000. An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. USA. 97:5978-83.