E.coli homologous recombination (recombineering) using Lambda Red for BAC backbone swapping

This protocol describes the steps required to swap a BAC backbone for a Big-IN or mSwAP-In backbone in the E. coli strain that the BAC is in. 

NOTE: lambda red (LR) phage homologous recombination is not a DNA repair mechanism such as with yeast HR! The mechanism is not entirely understood yet, but a key thing to know is that during recombineering, the lambda red system allows your donor DNA to essentially "invade" the target plasmid during normal REPLICATION - not repair. No cutting is involved. Recombineering can efficiently replace a region of interest with a donor fragment, but don't expect it to stitch together many fragments like in yeast.

Suggested reading: λ Recombination and Recombineering

Assembly design: We mainly use LR recombination to assemble a segment of a repository BAC into a delivery vector (e.g. one of our YAVs for Big-In or mSwAP-In). To this end, you first need to clone an "acceptor fragment" into your target vector. The acceptor fragment contains two homology/assembly arms, which we call LAA and RAA (left and right assembly arms). They are ~200 bps and will specify the termini of your assembled region. A unique RE site needs to be added between the LAA and RAA to allow efficient linearization of the acceptor vector prior to transformation. 

Step 1: CaCl2 heat shock transformation of Lambda red (LR) plasmid

Materials/Equipment required:

  • Cold 0.1M CaCl2

  • 12.5 mg/mL chloramphenicol aliquot

  • Lambda red plasmid DNA (pKBA13, a gift of Dr. Menghan Liu, Saeed Tavazoie lab@Columbia U)

  • BAC of interest (DH10B)

  • LB+carbenicillin (100 ug/mL) plates spread with chloramphenicol (25 ug/mL)

    • Add 50 uL 12.5 mg/mL chloramphenicol to LB+carb plate + 50 uL H2O and spread with beads. Let plate dry face-up at room temperature for 5-10 mins before use (if needed, can store at 4°C)

  • Pre-chill centrifuge (4°C)

Protocol:

  1. Grow BAC in 5 mL LB+chloramphenicol (12.5 ug/mL) overnight at 30°C

  2. Transfer 1 mL overnight culture to a 1.5 ml tube & chill on ice for 5 min

  3. Spin at 4000xg 1 min at 4°C

  4. Wash with 1 mL cold 0.1M CaCl2, spin 4000xg 1 min

    1. Repeat the wash, spin again

  5. Resuspend in 100 uL 0.1M cold CaCl2

  6. Use 50 ul each for:

    1. 10 ng lambda red (pKBA13)

    2. 1 ng pUC19

  7. Incubate on ice 20 mins

  8. Heat shock at 42°C for 45 sec, then ice 2 min, skip SOC recovery and plate total volume + 50uL H2O on LB+carb+chl plate

  9. Grow at 30°C ~20-24 hrs

    1. No need for colony PCR – you can just move forward with 5 mL overnight of 1 colony in LB+carb (100 ug/ml) + chloramphenicol (12.5 ug/mL), grow at 30°C

    2. Plate(s) can also be stored at 4°C for later use

Step 2: Preparation of BAC+LR-containing electrocompetent cells

Materials/Equipment required (for steps 2+3):

  • 20% L-arabinose solution (freshly prepared or stored at -20C)

  • 250 or 500 mL flask

  • 300 mL ice cold deionized H2O (per each assembly)

  • 2 mL tubes (cold)

  • 1000X carb aliquot (or pre-made bottle of LB+carb (100 ug/mL))

  • 12.5 mg/mL chloramphenicol aliquot

  • Ice bucket

  • Electroporation cuvettes, 0.2 cm (kept cold)

  • Linearized delivery vector with assembly arms (assembly arms must be on the plasmid, not as separate fragments)

  • LB+Kanamycin (50 ug/mL) plates

  • Pre-chill centrifuges (4°C)

Protocol:

  1. Grow 5 mL overnight culture of the strain containing pKBA13 +  BAC at 30°C in LB+carb(100 ug/mL) + chloramphenicol (12.5 ug/mL)

    1. Check the OD600 of overnight culture (diluted 1:20)

    2. Inoculate the overnight culture into 100 mL of LB+carbenicillin+chl (same concentrations as above) also containing 0.2% arabinose, to a final OD600 = 0.04)

    3. Grow at 30°C, 200-220 rpm for 3-3.5 hours (OD600 = 0.3-0.5)

  2. Pour culture(s) into 250 mL centrifuge tubes then place on ice 10 mins

    1. If you have many cultures, place flasks on ice until you can pour them into the centrifuge tube

  3. Spin down cells 4000xg 5 mins at 4°C

    1. Dump supernatant

  4. Wash the cells 3 times in an equal volume of cold (4°C, keep on ice during washes), deionized water (same speed/time as above) 

    1. Add 10 mL cold H2O with 10 mL serological pipet to break up pellet, then add 90 mL cold H2O

  5. After the third wash, resuspend cells using the remaining water in the tube after dumping supernatant. Then transfer all the cells to a new 2 mL cold tube.

  6. Spin 5000xg 1 min in small centrifuge at 4°C

    1. Dump supernatant

    2. Resuspend in 200-300 uL cold nuclease-free water

      1. Adjust amount of water relative to amount of cells - if they seem clumpy, add more water.

    3. Use 50 uL cells per sample

  7. Proceed to step 3 (It is perhaps possible to freeze the competent cells, but we've never tried it)

Step 3: BAC assembly with acceptor vector

  1. Electroporate DNA to the cells as you normally would for E. coli (Ec2 setting).

    1. 50 - 100 ng linearized delivery vector (digested overnight and gel/column purified)

      1. We have vectors (pWZ840, pKBA135 (MC2 v 2.0)) that are copy-number-inducible

      2. It is suggested to use Kanamycin for your delivery vector

      3. You can do multiple electroporations with the same linearized vector to increase colony number

    2. 1 ng undigested delivery vector as positive control

    3. no DNA as negative control (No need to electroporate the no DNA cells, just plate).

  2. Immediately add 1 mL SOC media to the cells and incubate for 1 hour in 30°C roller/shaker.

  3. Following SOC recovery, spin 5000xg for 1 min at room temperature, then resuspend cells in 100 uL H2O.

  4. Plate 100% on LB+Kan (50 ug/mL) plates and incubate at 30°C for 20-24 hrs

Assembly selection:

*Optional: Before PCR screening, you can replica plate the original plates to LB+Chl (25 ug/mL), and then to a new LB+Kan plate, and only choose Chl- colonies in screening. This also helps to purify the Kan colonies. Alternatively you can lose Chl+ colonies later when you replica plate to lose Carb+ colonies

  1. Choose a minimum of 11 colonies and do colony PCR with left/right junction primers (27 cycles, 55°C annealing). Include acceptor vector DNA to make sure your assays are specific enough - we have gotten false positives for the junctions before! 

    1. Choose 2-3 winners and grow overnight in 2 mL LB+Kan (50 ug/mL). If your payload is < 50 kb, you will have an easier time losing the lambda red plasmid growing overnight at 37°C. If it is larger than 50 kb, grow at 30°C.

      1. Please note that growing overnight in liquid Kan culture is essential to increase your chances of losing the lambda red (Amp/carb) plasmid!!! Your chances will be very low if you only do a single colony streak on a new plate, as you won't get as many colonies as plating by OD, and you are not getting the number of cell divisions that you will get in overnight culture.

  2. Take OD of 1:20 dilution of overnight culture and plate ~200 cells on LB+Kan (assuming OD=8 x 108cells/ml, link), incubate plates at 30°C overnight

  3. Replica plate on LB+carb (100 ug/mL), then LB+chl (25 ug/mL; if you haven't already replica plated to LB+chl), then a new Kan plate and incubate plates at 30°C overnight, and look for Kan+Amp-+Chl- clones.

  4. Choose 2-4 colonies and do colony PCR (27 cycles, 55°C annealing) with left and right junction, lambda red (oWZ2106/2107; ~1 kb), and chloramphenicol (oWZ2108/2109; 401 bp). Including internal assays here is optional - we have included them in the past with near 100% efficiency, but it is recommended to include at least 1 internal assay for payloads larger than 50 kb.

    1. Create a patch/mirror plate of winners on LB+Kan

    2. Remember to include Induction Solution in any liquid cultures you are growing for prep! (if you have an inducible vector)

Sequencing verification:

  1. We have seen instances of E. coli transposon (IS) insertions into the payload. Plasmidsaurus/Oxford Nanopore sequencing is highly recommended for ruling these out.

  2. If you do short reads Illumina sequencing, make sure you include all possible IS sequences in your reference, or implement bamintersect in your analysis.