Fosmid recombineering & Fosmid library

Fosmid recombineering (Kerri Spilker 10/5/09, based on Oliver Liu 7/6/09)

Reference: Tursun et al., 2009. A Toolkit and Robust Pipeline for the Generation of Fosmid-based Reporter Genes in C. elegans. PLOS One.

Notes:
• SW105 is always grown at 32ºC because the recombination proteins are induced at 42ºC.
• Make fresh electrocompetent SW105 cells to introduce fosmid because Oliver was not having luck with frozen competent cells. (see below)
• PCR template should be linearized before being used. (see below)
• Fosmids contain chloramphenicol resistance (Chlor).
• PCR product contains kanamycin resistance (Kan) flanked by LoxP sites.
* Arcing of the electroporator occurs when there is too much salt in the sample + DNA. Try using less DNA or more cells.

Grow up fosmid of interest
We purchased the entire fosmid library from Geneservice. You can get a copy of the Excel spreadsheet for the entire collection here:
http://www.geneservice.co.uk/products/clones/Celegans_Fos.jsp
Search the file to determine the location of your clone in the collection.
The 384-well plates are stored in the -80ºC freezer by the elevator. Use a tip to scrape some of the culture out. Streak on a Chlor-LB plate. Reseal the plate with foil tape and roller (kept in Oliver’s drawer.)
1. Grow fosmid strain from collection (in EPI300 at this point, ideally from a fresh colony) O/N at 37ºC in a 5mL culture of LB + 12.5mg/mL chloramphenicol.
2. Dilute into induction media: 4.5mL LB+chloramphenicol, 500uL of O/N culture, 5uL of 1000x Copy Control Induction solution.
3. Grow at 37ºC for 5 hours.
4. Perform column miniprep using miniprep columns. Be sure to elute using EB heated to 65ºC. Elute in 30uL.

Make electrocompetent SW105 recombineering strain and introduce fosmid
1. Grow 5mL O/N culture of SW105 in LB at 32ºC.
2. Dilute 1/50 in 10mL of LB.
3. Grow ~ 2.5 hours to OD600=0.6
4. Cool cultures on ice for a few minutes. Throughout this protocol, keep cells on ice.
5. Spin down at 4000K at 4ºC for 5 min. In the Sorvall 5CRC, 4000K is 2429 RCF. If you use the Luo Lab centrifuge, be sure to enter the correct speed.
6. Pour off supernatant. Add 1mL ice water. Gently pipette with P1000 a few times to mix. Add ice-cold water up to 10mL.
7. Spin down and repeat wash with 10mL of ice cold water.
8. Spin down and pour off supernatant (blot on a paper towel), leaving ~80-100uL left in tube.
9. Transfer 40-50uL to a pre-chilled eppie tube.
10. Use 1-2uL of fosmid to transform electrocompetent SW105 cells. Use 2mM cuvettes and 2.5kV standard protocol.

1. Transfer competent cells + DNA to a Bio-Rad Gene Pulser cuvette.
   b. On Luo electroporation machine: 4 (preset) – 1 (bacterial) – 2 (recombineering) – Hit Red “Pulse” button.
   c. After electroporation, add 1mL LB and transfer cells to a pre-chilled Eppendorf tube.
2. Recover 1 hour at 32ºC with shaking and plate on LB-Chlor plates.
   12. Incubate O/N at 32ºC.

   Making PCR product with homology arms for recombination
   1. Design PCR primers with 50 bp of homology to one side of the region of interest and ~25bp of the template that you wish to amplify. Purification of oligos is not necessary.
   2. Cut the recombineering template plasmid (pOL007, for example) and gel extract the appropriate band. If circular plasmid is used in the PCR reaction, it may contaminate the final PCR product and you will select SW105 cells that take up this Kan-containing plasmid.
   3. Amplify from the recombineering template using Phusion and a 50ºC annealing temp. Oliver has made many versions of the template.
   4. PCR purify the PCR product. (Important: Gel-purified fragments have historically not worked!)

   Amplified band should look like this:
   50 bp homology to insertion site + recombineering cassette (GFP::SL2::mCherry, for example) + 50 bp homology to insertion site

   Recombineering
   1. Grow 5mL O/N culture of fosmid-containing SW105 in LB-Chlor at 32ºC.
   2. Dilute 1/50 in 10mL of LB-Chlor.
   3. Grow ~2.5 hours to OD600-0.6.
   4. Make sure water bath is set to 42ºC. (Water bath on Oliver’s old bench.)
   5. Place tubes in 42ºC water bath for 15-20 minutes with occasional mixing. The recombineering proteins are being induced with this heat shock.
   6. Cool cultures on ice for a few minutes. Throughout this protocol, keep cells on ice.
   7. Spin down at 4K at 4ºC for 5 min. In the Sorvall 5CRC, 4000K is 2429 RCF. If you use the Luo Lab centrifuge, be sure to enter the correct speed.
   8. Pour off supernatant. Add 1mL ice water. Gently pipette with P1000 a few times to mix. Add ice cold water up to 10mL.
   9. Spin down and repeat wash with 10mL of ice cold water.
   10. Spin down and pour off supernatant (blot on a paper towel), leaving ~80-100uL left in tube.
   11. Transfer 40-50uL to a pre-chilled eppie tube.
   12. Add PCR product and electroporate. (Use 200ng, no more than 5uL). Notes: “Arcing” during electroporation is due to high salt. Wash cells again, use 100uL of cells, or reduce amount of DNA used.
   13. Recover 3 hours at 32ºC with shaking and plate on LB-Kan plates. Recovery is long to allow cells to divide more and increase the chance of finding a bacterial clone with one copy of the recombined fosmid.
   14. Incubate O/N at 32ºC.

   FLP-induction
   1. Grow O/N culture in 5mL LB-Kan at 32ºC.
   2. In the morning, spin down cells to remove LB-Kan and dilute 1/50 in 5mL LB+Chlor
   3. After ~2h, add 1/100 volume of 10% arabinose to induce Flp recombinase. Arabinose is stored at room temp on chemical shelves. Make 10% solution fresh. Sigma #A3256
   4. Incubate 2h at 32ºC with shaking.
   5. Spin down cells. Use miniprep solutions P1, P2, and N3 as usual.
   6. Spin 10min at max speed.
   7. Instead of using spin column, transfer 800ul of supernatant to new tube.
   8. Add 700ul of isopropanol. Mix (and store at -20ºC for 15min if you want).
   9. Spin down 5 min. and wash pellet with 500uL 70% EtOH.
   10. Spin down 5 min., aspirate dry. Allow to dry for at least 15 min., then resuspend in 50ul H20.
   11. Verify construct by PCR. (Example: PCR the entire recombinant region. Design primers outside of the squences used for recombination. Be sure the Kan(r) was excised.)
   12. Transform electrocompetent EPI300 with correct construct.

• Use 25uL of electrocompetent EPI300 cells (commercially available) + 2 uL recombinant fosmid.
• Electroporate with same conditions as for SW105.
• Recover for 1 hour at 37ºC in LB.
• Plate on LB-Chlor.
• Grow at 37ºC overnight.

13. Miniprep fosmid from EPI300 using typical fosmid miniprep (i.e. Copy Control Induction solution, columns and heated EB). Probably want to verify construct again by PCR to make sure you have plasmid that underwent FLP recombination.

    Inject fosmid at 1-4ng/uL