Bacterial Homing Endonuclease Selection System

Ryo Takeuchi 01/15/08

Technical notes for bacterial homing endonuclease selection system via gene elimination

(from Liu Lab)

Cloning

·  See the pEndo and pCcdB vector maps (pEndoSce.doc, pCcdTwt6.doc, pEndoAniI&pCcdBAniwtseq.doc).

·  AniI ORF in the pEndo plasmid is inserted between NcoI and NotI sites. The fMet of AniI is included in the NcoI site, and the stop codon is located at the 3’ terimnus of the ORF.

·  Incorporate 2 copies of the target site between AflIII and BglII (site 1), and between NheI and SacII (site 2).

- Prepare the ds oligonucleotides containing 2 copies of the target site with overhang restriction enzyme sites at both 5’ and 3’ side by annealing, and insert them into the pCcdB plasmid digested with the same restriction enzymes.

·  Use E. coli strain containing lacIq gene, (DH12S etc.) to get good yields with pCcdB plasmids.

Generate Electrocompetent cells harboring the pCcdB plasmid

1.  Transform the pCcdB plamid into DH12S competent cells, and grow the transformants on LB plates supplemented with 1 % glucose and 33 ug/ml chloramphenicol at 37 °C overnight.

- Autoclave the medium containing 1 % glucose for 15-20 min.

2.  Inoculate a few colonies in 10 ml of SBG media with 33 ug/ml chloramphenicol (3 % (w/v) tryptone, 2 % (w/v) yeast extract, 0.5 % NaCl, and 1 % glucose), and grow it at 37 °C overnight.

3.  Transfer all the overnight culture into 500 ml of SBG media supplemented with 33 ug/ml chloramphenicol, and grow the cells at 37 °C until OD reach 0.6-1.0 (It would take ~3 h).

4.  Put the flask containing the cells on ice for 15 min, and centrifuge them at 4,000 r.p.m. for 15 min at 4 °C.

5.  Resuspend the bacteria pellet in 200 ml of 1 mM Hepes-NaOH (pH 7.0) on ice, and centrifuge it at 4,000 r.p.m. for 12 min at 4 °C.

- Take the supernatant off by decantation and perform the following procedures below 4 °C.

6.  Resuspend in 100 ml of 1 mM Hepes-NaOH (pH 7.0) on ice, and centrifuge at 4,000 r.p.m. for 12 min at 4 °C.

7.  Resuspend in 50 ml of 1 mM Hepes-NaOH (pH 7.0) on ice, and centrifuge at 4,000 r.p.m. for 12 min at 4 °C.

8.  Resuspend in 20 ml of 10 % (v/v) glycerol on ice, and centrifuge at 4,000 r.p.m. for 10 min at 4 °C.

9.  Resuspend in 1 ml of 10 % (v/v) glycerol on ice, and dispense the cell suspension in tubes (50 ul each).

10.  Freeze in liquid nitrogen, and store at -80 °C.

Cleavage assay in bacteria

Before starting the assay, prepare the following materials.

·  10 x M9 salt

- Mix 60 g/L Na2HPO4, 30 g/L KH2PO4, 5 g/L NaCl, and 10 g/L NH4Cl.

·  1 M MgSO4, 1 M CaCl2

- Dissolve in water, and filter sterilize or autoclave for 30 min.

·  1 % thiamine, 100 mg/ml carbenicillin, 20 % L-arabinose, and 0.5 M IPTG

- Dissolve in water and fiter sterilize.

·  Positive selection and control plates

- Prepare 100 ml of 3 % (w/v) agar and 2 x M9 salt-based solution (2 x M9 salt, 2 % (v/v) glycerol, and 1.6 % tryptone) separately, and autoclave for 30 min. Mix both the solutions, and then cool down below 60 °C. Add 200 ul of 1 M MgSO4, 1 M CaCl2, 100 mg/ml carbenicillin and 40 ul of 1 % thiamine. Add 200 ul of 20 % L-arabinose and 160 ul of 0.5 M IPTG for the positive selection plates.

·  2 x YT media

- Mix 16 g/L tryptone, 10 g/L yeast extract, and 5 g/L NaCl and autoclave for 30 min.

1.  Keep 1 ml and 1.6 ml of 2 x YT media (per cuvette) at 42 °C and at RT, respectively.

- Use the former media to recover the transformants, and the latter to supply carbenicillin and L-arabinose.

2.  Transform 1 ul of the pEndo plasmid (0.1-1 ng/ul) into 50 ul of the DH12S competent cells harboring the pCcdB plasmid by electroporation.

- I conducted an electroporation in 0.2 cm cuvettes using Gene Pulser (Bio-Rad) with 2.5 kV.

3.  Immediately, add 1 ml of 2 x YT media (warmed at 42 °C) into the cuvette and mix by pipetting.

4.  Transfer 400 ul of the cell suspension into a round-bottle tube, and shake at 37 °C for 30 min.

5.  Add 1.6 ml of 2 x YT media supplemented with 125 ug/ml carbenicillin and 0.025 % L-arabinose, and grow at 30 °C for 4 h.

- Expression of the homing endonuclease for 4 h is necessary before plating. Otherwise, the survival rate would be decreased.

6.  Centrifuge 1 ml of the culture at 5,000 r.p.m. for 5 min at RT, and resuspend the bacteria pellet in 600 ul of sterilized water.

7.  Spread 50-100 ul of the cell suspension on both the positive selection plates and the control plates.

8.  Incubate the plates at 30 °C for ~36 h.

9.  Count the number of the colonies, and calculate the suvival rate on the selective plates: [Survival rate (%)] = [Colonies on the selective plates] / [Colonies on the control plates] x 100.

Large-scale selection

1.  Transform 50-100 ng (1-4 ul) of the pEndo library into 50 ul of the DH12S competent cells harboring the pCcdB plasmid by electoroporation, and immediately add 1 ml of 2 x YT media (warmed at 42 °C) into the cuvette.

2.  Transfer all the cell suspension into a 50 ml conical tube, and shake at 37 °C for 30 min.

3.  Add 4 ml of 2 x YT media supplemented with 125 ug/ml carbenicillin and 0.025 % L-arabinose, and grow at 30 °C for 4 h.

4.  Centrifuge at 4,000 r.p.m. for 10 min at RT, and resuspend the bacteria pellet in 1 ml of sterilized water.

5.  To calculate the transformation efficiency, dilute 2 ul of the cell suspension with 200 ul of sterilized water, and spread 100 ul of the dilution on the control plate.

6.  Spread 500 ul of the cell suspension on 150 mm petri dishes containing the positive selection media.

7.  Incubate the plates at 30 °C for ~36 h.

8.  Transfer the colonies on both the positive selection and the negative selection plates, and incubate at 30 °C overnight.

- The negative selection plates contain almost the same chemicals as the positive selection plates, but include 33 ug/ml chloramphenicol instead of 0.4 mM IPTG.

9.  Amplify I-AniI genes on the pEndo plasmids by direct PCR from clones surviving only on the positive selection plate.

- I used the following primers; 5’- CAC GGC AGA AAA GTC CAC ATT G -3’ and 5’- TGA GGG AGC CAC GGT TGA TG -3’. I performed 30 cycles of PCR amplification (95 °C for 30 sec, 50 °C for 30 sec, and 68 °C for 1 min 5 sec) in 20 ul of the reactions with AccuPrime Taq DNA polymerase (Invitrogen).

10.  Check the PCR products by electorphoresing 1 ul of the reactions on an agarose gel.

11.  Mix all the PCR products, and purified with Qiagen PCR purification kit.

12.  Digest 1-5 ug of the purified PCR products and the pEndo plasmid with 1 ul of NcoI and Not I at 37 °C overnight.

13.  Purify them by gel extraction, perform the ligation, and purify with Qiagen PCR purification kit (elute with 30 ul of TE).

14.  Transform 2-4 ul of the ligated DNA into 50 ul of electrocompetent cells, and spread the cells on LB plates containing 50 ug/ml ampicillin after incubation in LB or SOC media at 37 °C for 30 min. Incubate at 37 °C overnight.

15.  Scrape off all the colonies from the plates, and grow at 37 °C for a few hours through overnight.

16.  Purified the plasmid with mini prep, and trasform <1 ng of the plasmid into 50 ul of the DH12S competent cells harboring the pCcdB plasmid.

17.  Immediately, add 1 ml of 2 x YT media (warmed at 42 °C) into the cuvette and mix by pipetting.

18.  Transfer 400 ul of the cell suspension into a round-bottle tube, and shake at 37 °C for 30 min.

19.  Add 1.6 ml of 2 x YT media supplemented with 125 ug/ml carbenicillin and 0.025 % L-arabinose, and grow at 30 °C for 4 h.

20.  Centrifuge 1 ml of the culture at 5,000 r.p.m. for 5 min at RT, and resuspend the bacteria pellet in 600 ul of sterilized water.

21.  Spread 50-100 ul of the cell suspension on both the positive selection plates and the control plates.

22.  Incubate the plates at 30 °C for ~36 h.

23.  Repeat the procedures 8-10.

24.  Purified the PCR products separately with Qiagen PCR purification kit.

25.  Sequence each PCR product.

- I used the 130-bp upstream primer of I-AniI ORF (5’- CGG CGT CAC ACT TTG CTA TG -3’) for sequencing.