Protocol Code: CT6

Conjugal transfer into Anabaena

(Taken from Jeffrey Elhai Lab)

Notes

This protocol is designed for the routine transfer of plasmids into Anabaena PCC 7120. If you are looking for a protocol that gives quantitative results, see Elhai et al (1997) [J Bacteriol 129:1998-2005].

Two methods are described, spot mating (useful when exconjugants figure to be common, e.g. with plasmids that can replicate in Anabaena) and plate mating (useful when exconjugants figure to be rare, e.g. with plasmids that can't replicate in Anabaena)

Both methods are described further in Elhai & Wolk (1988) [Methods Enzymol 167:747754] and Elhai et al (1990) [Plant Molec Biol Manual A12:1-23].

Raw Material

• Donor strain(s): E. coli strain[conjugal,helper,cargo]
  • (example) HB101[pRL443(ApTc),pRL545(Km),pRL623(Cm)] or HB101[pRL443(ApTc)] (conjugal plasmid) + HB101[pRL545(Cm) (cargo),pRL623(Km) (helper)]
• Recipient strain: Anabaena PCC 7120
• Mating plates: BG11 or BG11 + 5% LB (~15 ml in 6cm plate)
  • Need one dish per mating
• Selection plates:
  • Select for recipient (by phototrophic growth on BG11)
  • Select for exconjugants with cargo (BG11 + antibiotic)

• Selection filters: nitrocellulose, detergent free, sterilized

Time Line

DAY 0: Unfreeze E. coli, start Anabaena, make plates

1. STREAK OUT DONOR STRAINS onto selective plates. Add one appropriate antibiotic per plasmid. Refrigerate plate after overnight growth. Good for a week or two.
2. GROW UP RECIPIENT Anabaena in BG11. Aim for 50 ml of late log phase culture by Day 2. Figure one doubling every 12 hrs under optimal conditions, i.e., growth at 300 and light around 50 μEinsteins.
3. PREPARE PLATES: Mating plate = BG11 + 5% LB; Selection plate = BG11 + antibiotic.

DAY 1: Grow E. coli

1. GROW UP DONOR E. coli STRAINS in LB + antibiotics. Don't select for chromosomal markers. 3ml culture is sufficient. Grow in 37° shaker overnight.

DAY 2: Start mating

1. REGROW DONOR E. coli in LB, inoculated 1:20 with overnight culture. Grow at least 2 ml of each strain per plate mating or 2 ml total for spot matings, about 2 hours.
2. HARVEST REGROWN E. coli STRAINS, spinning down 1.5 ml of each strain in a microfuge tube. Spin down 30 sec in microfuge. Remove all supernatent.
3. WASH E. coli STRAINS. Resuspend pellets in 1 ml of LB, by pipetting up and down. Do not vortex. Pellet in microfuge. Remove supernatant. If there are two donor strains in a conjugation (conjugal strain plus helper/cargo strain), then resuspend the pellets as before in 500 ul, mix the two suspensions, centrifuge, and remove supernatant. The pellets can sit while you harvest Anabaena.
4. HARVEST Anabaena. Pellet culture in sterile 50 ml centrifuge tubes. Transfer pellets to microfuge tube(s). Spin down, and resuspend in volume roughly equal to twice the volume of the pellet.
5. PREPARE MATING PLATE by placing a sterile mating filter on a BG11+5%LB plate. Avoid air bubbles.

Plate mating

1. DILUTE Anabaena. In a separate tube, make a 1:10 dilution of the concentrated Anabaena suspension.
2. MIX E. coli DONORS AND Anabaena RECIPIENTS in microfuge tube. Prepare two tubes per mating. To each tube add half of the concentrated E. coli, plus either the concentrated or diluted Anabaena, adjusting the total volume to ~200 μl with BG11.
3. SPREAD ON MATING PLATES, transferring each E. coli /Anabaena mixture to one half of the filter on the mating plate, spreading by tilting the plate and painting the filter with a pipet tip (sucking up and down). Don't use a glass spreader. Let the filter dry in hood.
4. INCUBATE MATING PLATES, 30° in light box for specified length of time (24-48 hrs). Put plates top up or top down, depending on the incubator, so that condensation does not accumulate on the top of the Petri plates. Do not stack the plates (so that they all see the same amount of light).

Spot mating

1. DILUTE Anabaena. Serially dilute the dense cyanobacterial suspension 1:10, up to 1:106 in any convenient volume of GBG11. For each dilution you will need 5 μl per mating.
2. MIX E. coli DONORS AND Anabaena RECIPIENTS. Add 50 μl of L broth to one E. coli mixture (Step C) and mix with a pipette. Apply a 5 μl spot of each dilution of Anabaena to an empty sterile Petri dish. To each spot apply 5 μl of the E. coli mixture, mix well, and transfer 2 μl to the filter in some logical grid. Repeat this step for each E. coli mixture. Let the filter dry in hood. It's a good idea to include as a control a dilution series of either Anabaena alone or (better) Anabaena mixed with E. coli lacking conjugal plasmid.
3. INCUBATE MATING PLATES, 30° in light box for specified length of time (24-48 hrs). Put plates top up or top down, depending on the incubator, so that condensation does not accumulate on the top of the Petri plates. Do not stack the plates (so that they all see the same amount of light).

DAY 3 or 4 (may vary): Transfer mating to selective media

1. TRANSFER FILTER TO SELECTION PLATE. Prewarm the plate to 300. Be sure to avoid air bubbles underneath the filter.
2. INCUBATE SELECTION PLATES, about a week at 30° under light.

DAYS 5-11 (may vary): Monitor conjugation

1. MONITOR SELECTION PLATES. The amount of green should gradually die away. If you observe that background growth continues after transfer to the selection plate, it might be possible to rescue the conjugation by transferring the filter to a higher level of antibiotic.
2. GROW EXCONJUGANTS. Grow apparently antibiotic-resistant colonies in BG11 + appropriate antibiotic. Especially when using C.K3 as the selectable marker, fake resistance is not uncommon, but colonies that have not stably integrated the plasmid will not grow in liquid plus antibiotic.