ProcedureGenomeModificationDatsenkoWanner < Lab

---++ Lambda Red Protocol
 
---++ Lambda Red Plasmids

These plasmids are available as part of the [[http://cgsc.biology.yale.edu/GDK.php][Wanner Lambda Red disruption kit]] from the [[http://cgsc.biology.yale.edu][<i>E. coli</i> Genetic Stock Center]].

   * pKD46 (ampR)
   * pKD78 (camR)

---++ Making Competent Cells expressing Lambda Red

---+++ Before Day 1
   * Transform your strain of interest with pKD46/pKD78 like you would with any plasmid.  Recover at 30°C for 1h 30min to 2h.
   *  Recovery can be done for 1H at 37°C if trying to save time.
      * This is because recovery does not require plasmid replication, only expression of the antibiotic resistance cassette.
   *  After recovery, plate on appropriate media and grow overnight at 30°C.

---+++ Day 1

   * Grow starter culture (of strain of interest with pKD46/pKD78) in 10mL LB-Amp or LB-Cam overnight at 30°C.

---+++ Day 2

   * Add 500uL of overnight culture to 30mL LB-Amp (or Cam).
   * Grow on 30C shaker until OD600 = ~ 0.350 - 0.400, (takes ~2-3 hours)
   * Induce cultures with filtered 10% Arabinose, creating a final concentration of 0.1% Arabinose (300uL of 10% Arabinose into the 30mL of culture).
   * Place back on 30°C shaker for 15 minutes, then transfer to 50mL falcon tube, and place on ice for 40 minutes.  Start chilling centrifuge to 4°C.
   * Spin cultures @ 1800 x g for 15 min to pellet cells.  
   * Resuspend pellet in 30 mL cold water.
      * cold 10% glycerol is also commonly used instead of water without incident.
   * Spin @ 3000 x g for 5 minutes, resuspend in cold water.
      * cold 10% glycerol is also commonly used instead of water without incident.
   * Spin @ 3000 x g for 5 minutes, and resuspend in cold 10% glycerol.
   * After this spin cycle, while pouring out the glycerol pay close attention to the pellet.  It is likely somewhat loose/mushy at this point, so pour off the glycerol until the pellet starts to move, then briefly vortex to resuspend the entire pellet, and transfer the entire remaining volume to a cold 15mL tube.  Remaining volume will likely be between 10 to 3 mL.
   * Rebalance centrifuge, spin @ 3000 x g for 5 minutes.  Pour off all glycerol, pellet should be stable.
   * Add back cold 10% glycerol for desired cell density, and number of elctroporations.  Usually 300uL will yield a good cell density for ~6 electroporations.  Resuspend pellet by pipetting with 1mL pipette.
   * Aliquot 50 uL cell suspensions into cold eppendorf tubes, store at -80°C.

---++ Transforming Cells

   * Add DNA to cell aliquots, typically 50-200 ng (be sure to desalt, or use very low volumes of DNA i.e., 1uL)
   * Transfer cells + DNA to a chilled gap cuvette
   * Electroporate, recover immediately with 1mL SOC/SOB/LB.
   * Transfer to a culture tube with 10mL SOC/SOB/LB, place on 30°C shaker for 1.5 hrs.
      * The 1ml recovery solution can directly be placed at 30°C shaker for 1.5 hrs (without adding it to 10ml as indicated)
   * Plate 100-200 uL of recovery onto selection plate (such as LB-Kan if using the Kan cassette from the keio collection). Leave the recovery culture on bench overnight, plate more next day if the initial plating was unsuccessful.
      * Can (and possibly should) store at 4&deg;C overnight rather than on bench.
   * Grow the selection plate at 37°C overnight; Colonies may take a full 24 hours to appear.

*NOTE* Recover at 30°C if you intend on maintaining pKD46.  This plasmid's origin is temperature sensitive and does not function properly at 37&deg;C.  Your insert will still recombine if recovering at 37°C.

---+++ Expected Results

The data below used 20 ng of purified PCR product targeting the kan cassette from the topB keio strain, and 50uL of lambda-ready cells.  To generate the insert, run a 30 µL PCR using the primers below and genomic DNA from the topB keio strain under standard PCR conditions (55&deg;C annealing temperature, 25 cycles).  Gel purify the 900 bp band.

*Primers*

SWS_19: topB forward, GAGGTCAAAGCTACAGCCGCC

SWS_20: topB reverse, ATACTTCTCGCTCCCAGGATGG

Location: Gottel stock primers, -20C freezer in MBB.

*Strain*: the topB keio strain is located in well P14 of the "33,35,37,39" plate in the keio collection rack, -80°C freezer in MBB.


*REL606*
%TABLE{headeralign="left,center,center,center,center,left" dataalign="center,center,center,center"}%
| *Colonies on selective plate* | *Colonies on control plate* | *Dilution Factor* | *Recombination Efficiency* |
| 5 | 65 | 10^5 | 7.7E-8 | |
| 8 | 37 | 10^5 | 2.2E-7 | |
| 7 | 53 | 10^5 | 1.3E-7 | |

*BW25113*
%TABLE{headeralign="left,center,center,center,center,left" dataalign="center,center,center,center"}%
| *Colonies on selective plate* | *Colonies on control plate* | *Dilution Factor* | *Recombination Efficiency* |
| 1 | 135 | 10^5 | 7.4E-9 | |
| 1 | 152 | 10^5 | 6.6E-9 | |
| 3 | 125 | 10^5 | 2.4E-8 | |


This method can also be used to edit plasmids in-vivo, especially when the plasmid is large and difficult to modify through other methods. The procedure is identical to the method used for chromosomal recombination. 50-100ng of purified PCR product was used to recombine with a medium copy 40kb plasmid successfully (10-15 copies). Recommended to plate the whole 1ml recovery mixture (spin it down, discard 900ul and resuspend the pellet in 100ul of media). Plate on selective media. Note that at this point cells will contain a mixture of both modified and unmodified plasmid. Grow colonies in selective liquid media and miniprep the cultures. This miniprep will also likely contain a mixture of plasmids. However, transforming the plasmid mixture and plating on selective plates should yield colonies that only contain the modified plasmid.


---+++ References

   1 Datsenko, K.A., Wanner, B.L. (2000). One-step inactivation of chromosomal genes in _Escherichia coli_ K-12 using PCR products. _Proc. Natl. Acad. Sci. USA_ *97*:6640-6645. <br> *Original paper describing method.*
   2 Mori, H., Baba, T., Yokoyama, K., Takeuchi, R., Nomura, W., Makishi, K., Otsuka, Y., Dose, H., Wanner, B. L. (2015) Identification of essential genes and synthetic lethal gene combinations in _Escherichia coli_ K-12. _Methods Mol. Biol._ *1279*: 45–65. <br> *Detailed protocol describing the construction of the Keio collection via the Datsenko & Wanner method.*

---+++ Contributors

   * Originally from Erik Quandt (6/7/2011)
   * Edited by Steven Sowa (6/2011)
   * Edited by -- Main.NeilGottel - 11 Sep 2012
   * Edited by DED (4/3/13) -- comment on use of glycerol rather than water after conversation with Lindsey and Mike
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Topic revision: r11 - 2025-07-16 - TylerDeJong