(r18) ProcedureGeneReplacements < Lab

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---+++ Allelic exchange using pKOV vector or modified variants.


---++++ Before beginning part 1: design primers
   1 Insert should be ~1kb with approximately 500bp on either side of mutation, without disrupting neighboring genes. 
   1 At 5' ends of both forward and reverse primers, include 1) short CG clamp (CGC), 2) NotI site (GCGGCCGC) on forward primer and BamHI restriction site (GGATCC) on reverse. So each forward primer should look like: 5'- CGC GCG GCC GC(normal PCRing primer) -3' and the reverse primer should look like: 5'- CGC GGA TCC (normal PCRing primer) - 3'
   1 Make sure amplified region does not contain NotI or BamHI sites (alternative sites can be used SmaI (CCCGGG) for forward, and SalI(GTCGAC) for reverse)
   
---++++Before beginning part 2: verify pKOV markers (ts, sacB, cat)
   1 Grow up overnight REL606 pKOV in LB + cam (tests camR)
   1 Dilute in saline 3x 1:100 (so 100ul in 10ml saline) and plate 100 ul on 1) LB cam @ 30 degrees, 2) LB cam @ 43 degree, and 3) LB + sucrose (no NaCl) @ 30 degrees. Cells should grow at 30 but not at 43 or with sucrose (or at least very few colonies). 
   1 Also make a glycerol stock of the overnight culture (200µl 80% glycerol, 1ml cells) and store in -80°C
   1 ALL remaining cells from the overnight culture should be spun down at 10,000xg for 10 minutes and miniprepped. You'll need your own plasmid stock for the BamH1 digestion. 

---++++ Inoculate strain (day 0)
   1 Grow up appropriate stain with desired mutation (and WT for a control) in 5ml LB overnight

---++++ Purify genomic DNA
   1 Use Invitrogen gDNA isolation kit. Spin down all 5ml of cells and follow kit instructions. Elute with 100ul water. 

---++++ PCR out gene
   1 PCR out desired gene with primers design as described above. Run multiple reactions for each gene (150-300 µL total) and pool the reactions to ensure there will be sufficient product left after subsequent purifications.
   1 Run gel with 5µl sample to verify PCR worked (if whole cell PCR doesn't work, gDNA extract and repeat the PCR. This should be reliable.)
   1 PCR purify rest of sample (It helps to elute DNA in 40µl for digestion step)
   
---++++ Not1/BamH1 double digestion

*You'll need to digest both vector plasmid and PCR insert(s). 
  
%TABLE{dataalign="center"}%
| *Reagent* | *Final concentration* | *µl* |
| Buffer 4 | 1X | 5 |
| BamH1-HF | 50 U | 2.5 |
| Not1-HF | 50 U | 2.5 |
| BSA | 1X | 0.5 |
| DNA | all of it | 39.5 |
| *total* | *-* | *50* |

   1 Digest 3hr at 37°C (For the HF restriction enzymes, you do not need to do a heat inactivation). 
   1 Store at -20°C or immediately run entire sample on gel for gel extraction.
   1 Gel extract both inserts (~1kb) and vector (the gel should have two bands for pKOV: one for the 3kb stuffer sequence and the larger 5.6 kb band being the doubly digested pKOV. A third, faint band may also be visible at 8.6 kb, which is the singly digested pKOV. Only the 5.6kb band should be gel purified, the other band(s) is discarded.) For the vector, a 0.5% gel should be used for better separation of the larger bands (0.250g agarose, 50mL buffer). 
   1 Elute gel extracted vector in 44µl water for the AP treatment step.
   1 Store purified DNA at -20°C or move on to AP treatment. 

---++++ Antarctic phosphatase treatment 

*This is a VECTOR ONLY step! Save your insert(s) for ligation.*

| *Reagent* | *µl* |
|cut vector | 44 |
| AP buffer | 5 |
| AP enzyme | 1 |
| *total* | *50* |

   1 Digest 15 minutes at 37°C, then 5 minutes at 65°C
   1 Ethanol precipitate the AP-treated vector and the digested insert(s) before ligation

---++++ Ligation

   1 Nanodrop (or Qubit) both inserts and vector and record ng/ul
   1 Look up the size (in bp) of both insert and vector. You'll need both these to calculate ligation volumes because concentration is based on molarity. 
   1 Calculate insert and vector volumes (insert will be 5x the molar concentration...see the formula below)

*Insert:*    (50/3000)(size in bp) = # / (concentration in ng/ul) = *x µl of insert*

*Vector:*   (10/3000)(size in bp) = # / (concentration in ng/ul) = *y µl of vector*

*Ligation Reaction*

| *Reagent* | *µl* |
| ligation buffer | 4 |
| insert | 2x |
| vector | 2y |
| T4 ligase | 2 |
| water | up to 40 |
| *total* | *40* |

Your negative control should be (cut) vector without an insert (add more water to make 40µl).

Incubate at 16°C overnight. Ethanol precipitate before transforming.

---++++ Transformation

   1 Thaw electrocompetent cells on ice (2 tubes for controls and 1 tube for each reaction)
   1 Put cuvettes on ice and thaw ligation product (plus negative vector-only control)
   1 Thaw uncut pKOV (this is your positive control)
   1 Add 1 to 3 µl of each DNA condition to 40µl REL606 cells, flick to mix and pipette into cold cuvettes. 
   1 Add 1ml room temperature SOC into the old microfuge tubes. 
   1 Electroporate each and immediately pipette 20µl (using red tips) room temperature SOC buffer into cuvette and pipette out all liquid from cuvette into the 1.5ml microfuge tube. 
   1 Incubate tubes shaking on their sides for overnight at 30°C to recover.  
      2. Alternatively, it should be OK to recover at 37°C 1-2H.  Plasmids will not replicate, but antibiotic resistance markers will be expressed none-the-less.
   1 Spin down the entire culture (15 min @ 3500 rpm), remove 800µl of the supernatant, and resuspend cell pellet in the remaining 200µl of supernatant. Plate 100µl onto two LB + cam plates (plating all 200µl on one plate will be too wet) onto LB + cam plates and grow overnight at 30°C. 

---++++ Confirming plasmid

   1 Use primers CKO3 = 5'-AGGGCAGGGTCGTTAAATAGC-3'  and EKO33 = 5'-TTAATGCGCCGCTACAGGGCG-3' to screen camR colonies (the two are on the plasmid, it will say if there is an insert of the right size or not in the vector)
   1 PCR n°2 on 2&#956;L cultures (for clones ok with the PCR n°1) with CKO3 and the primer 1 of the first cloning step, another with CKO3 and the primer 2 of the first cloning step (CKO3 is on the plasmid, primer 1 and 2 on the insert but you don’t know what is the sense of the insert on the plasmid. It will say if there is an insert the one you are expected)
   1 If OK, make a plasmid maxiprep on 1 or 2 colonies.

---++++ Allelic exchange with suicide plasmid pKOV

   *-Permissive T° : 30°C*
   *-Restrictive T° : 43°C*
   1 Transform desired electrocompetent strain (REL606, MG1655, etc) with 100-500ng of suicide plasmid.
   1 Incubate for 2-5 hr at 30°C to be safe.
      a. To save time, 45 min at 37°C and 45 min at 30°C should be fine too.  If intending to directly select for integrants, leave them to grow at 30°C for an additional hour.
   1 Plate cells on LB agar + cam plates and grow overnight at 30°C.
      a. *Alternatively, you can attempt to directly obtain integrants and save a day's work.* This is what the Church lab suggests on their protocol.
         i. Divide transformed cells onto two separate plates. Grow one overnight at 30°C and and the other 43°C. This allows for measuring integration frequency (# integrants at 43°C/ # transformants 30°C) and also ensures that you will at least get plasmid transformants.
         i. If integrants are obtained, skip to step 9.
         i. If no integrants are observed, then use transformants from the 30°C plate and continue with this protocol.
   1 Pick ~6 colonies and dilute in 200µl saline. 
   1 Dilute this 1:10, 1:100, and 1:1000 in 200µl saline. Spread 100µl of these dilutions on LB + cam plates (preheated at 43°C). *Integration of the plasmid step.*
   1 Grow these plates overnight at 43°C. 
   1 In addition, streak once directly from the 30°C plate to another 43°C LB + cam plate (reduces noise of the temperature resistant colonies). 
   1 Lastly for the integration step, save 4 transformant colonies from the LB + cam 30°C plates in 40% glycerol.
   1 Next day, pick a colony without taking satellites and dilute in 1ml saline. Dilute this another 1:10, 1:100, and 1:1000 in 200µl saline. 
   1 Plate 100µl of all dilutions (including the 1ml "undiluted") on LB (no NaCl) + 5% w/v sucrose plates. Incubate these at 30°C overnight. *Excision of the plasmid step.*
   1 Replica plating onto LB + cam plates: patch 50 to 200 colonies to LB + cam plates and grow overnight at 30°C. (If the 1:1000 plate had more than 10 colonies, something is wrong... toss these and try again). Note: Loss of camR should be 90-100% for the colonies picked. *Testing for loss of plasmid (camR)*
   1 Recover the same 50 to 200 clones in LB and 20% glycerol and store at -80°C. 
   1 Screen clone for the desired allele PCR and sequencing. (First genomic DNA isolation if whole cell doesn't work?)

---++++ References

   1 Link, A. J., Phillips, D., and Church, G. M. (1997) Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization. _J. Bacteriol._ *179*:6228–6237.
Topic revision: r18 - 2025-02-19 - TylerDeJong