M13 Phage

== M13 Amplification Protocol == Grow overnight culture of F'+ E. coli (e.g. ER2738). Inoculate a 20 ml culture in a 250 ml Erlenmyer flask with 200 µL overnight E. coli culture. Add 1 µL phage suspension. Shake flask at 37°C, 250 rpm for 4 -5 hrs. Remove cells by centrifugation at 4500 g for 10 min. Transfer supernatant to a fresh tube. Repeat centrifugation. Transfer top 16 ml of supernatant to a new tube and add 4 mL of 2.5 M NaCl/20 % PEG-8000 (w/v). Briefly mix. Precipitate phage for 1 hr or overnight at 4°C. Pellet phage by centrifugation at 12000 g for 15 min. Decant supernatant. Resuspend pellet in 1 mL TBS. Transfer to an eppendorf tube. Spin briefly to remove any cell debris. Transfer supernatant to a fresh tube. Add 200 µL of 2.5 M NaCl/20% PEG-8000. Incubate on ice for 15-60 min. Spin 12000 -14000 rpm in a benchtop centrifuge for 10 min. Discard supernatant. Spin again briefly and remove remaining supernatant with pipette. Resuspend pellet in 200 µL TBS. For long-term storage at -20 °C, add 200 µL sterile glycerol. To scale up the above protocol, use multiple culture flasks. Alternatively, after incubating 20 ml culture for 2 hrs, add the entire culture to 1L LB. Incubate the large culture for 4 hrs, then modify the protocol to remove cells and purify phage. == Preparation of bacterial cells == 1. Pick a single colony from a freshly streaked plate with the phagemid-containing bacteria and inoculate a 3-ml culture of 2xYT medium supplemented with ampicillin 100 µg/ml and glucose 1% w/v. In absence of IPTG and presence of glucose 1% w/v, the expression of the g3p fusion protein is repressed; this helps preventing loss of phagemid or selection of insertless clones, and improves library integrity. 2. Incubate o/n at 37ºC with agitation at 250 rpm. 3. In the morning, dilute an aliquot of the culture 1:20 v/v with fresh 2xYT medium in a new culture tube and incubate for one hour at 37ºC with agitation at 250 rpm. It is important to dilute the glucose to 0.1% or less to prevent the catabolite repression of the lac operator.  4. Take a small aliquot in sterile conditions and measure the absorbance at 600 nm of a 1:10 dilution in 2xYT medium (A600). With SS320 or TG1 strains, absorbance should be close to 0.5 OD and cells are ready to be transduced. If necessary adjust the length of incubation to your conditions.  == Helper phage transduction == We recommend incubating directly at 37ºC with agitation at 250 rpm for 30 min to one hour after adding the helper phage. The total number of transductants is not increased by either pre-incubating bacteria without agitation for 10 min prior to adding the helper phage (sometimes said to regenerate pili destroyed by strong agitation) or after adding the phage (sometimes said to promote transduction). In fact we have noted that in our conditions a lack of agitation during the transduction period results in smaller number of transductants and variable virion production. We also recommend adding the helper phage when the bacterial culture reaches an optical density at 600 nm between 0.4 OD and 0.5 OD; large amounts of non-superinfected cells may decrease virion production above 0.5 OD while disparities caused by differences in phage growth rates will be amplified at lower ODs. 1. Optionally vortex vigorously M13KO7d3 and collect with a brief microcentrifugation; this treatment will disrupt potential phage aggregates and is indicated when reproducible and precise quantities of helper phage are desired. 2. Add 1 µl of M13KO7d3 phage per 1 ml of bacterial culture with A600 at 0.5 OD. This corresponds to a multiplicity of infection (MOI) around 14. 3. Incubate for 1 h at 37°C and 250 rpm; some protocols only recommend 30 min incubation for this step. 4. Add ampicillin 100 µg/ml, kanamycin 50 µg/ml, and IPTG 200 µM; lower the temperature to 30ºC and incubate 8 h to overnight before proceeding to phage purification. The amount of IPTG to add depends on the phagemid, e.g. phagemids such as pADL-23c, pHEN2 or pComb3 do not require IPTG while pADL-10b does require IPTG. The production of virions using helper phage is usually straightforward.  Insure the presence of pili by applying selective conditions during the growth of bacterial stocks (streak TG1 bacteria on M9-minimal medium plates) or during the culture itself if maintenance of the F’ episome is mediated by antibiotic resistance, e.g. tetR phenotype for SS320 (add tetracycline 50 µM to the growth medium).  Streaking a kanamycin-containing plate before adding M13KO7d3 may reveal contamination by another helper phage.  Lack or low virion production when bacterial cultures are too dense at the time of transduction. This problem is amplified by immunity to superinfection induced by endogenous expression of full-length g3p fusion protein by the phagemid. Make sure to only use bacterial cultures with A600 equal or inferior to 0.5 OD when adding the helper phage.  Phagemid whose phage coat protein fusion expression is not properly regulated may lead to toxicity and arrest of cell division as soon as the glucose is removed from the culture medium. Therefore a culture not dense enough when the helper phage is added may produce very few virions. Always use freshly prepared bacteria and a culture to the recommend density value to maximize phage yields. == Helper Phage == Storing the Helper Phage:  helper phages in 7% DMSO and recommends storing them at -80o C. As long as there is a –80o C stock in the lab, the amplified lab prep can be stored at +4o C. Titering the Helper Phage: Streak out E. coli strain XL1-Blue MRF′ (TG1) onto an LBTet15 plate. Pick a single colony of XL1-Blue MRF′, inoculate into LB and grow to OD600 = 1.0. (No need to include the antibiotics in the overnight cultures.) Meanwhile prepare a serial dilution of the phage in TE buffer. Expected number of phage should be about 1010 pfu/ml; therefore dilute phage to create dilutions 10-4 to 10-7. Note: When preparing serial dilutions of bacteriophage, it is usually much more accurate to dilute 10µl into 100 to create a 10-1 dilution or 10µl into 1000µl to create a 10-2 dilution rather than to dilute 1 into 10µl or 1µl into 100µl because one tends to obtain grossly exaggerated titers resulting from small pipetting errors. This is particularly critical for the first dilution. Add 200µl of XL1-Blue MRF′ cells at OD600 = 1.0 to individual sterile culture tubes (the number of phage dilutions you plan to plate) in a test tube rack. Add 100µl of each serial dilution of helper phage to the culture tubes containing the XL1-Blue MRF′ cells. Place the test tube rack into a 37o C water bath for 15 minutes to allow the helper phage to attach to the cells. Meanwhile melt NZY Top Agarose in the microwave and allow it to cool to ~48/50o C. You need to pay close attention to the bottle containing the top agarose because it can quickly boil over; alternatively use a low power setting on the microwave. {| border="1" cellspacing="1" cellpadding="1" style="width:500px;" |- |
| rowspan="1" colspan="5" | Dilutions
|- | Phage
| 10µl (stock) 
| 10µl (10^-2)
| 100µl (10^-4) 
| 100µl (1^0-5)
| 100µl (10^-6)
|- | TE
| 990µl
| 990µl
| 900µl 
| 900µl 
| 900µl 
|} At the conclusion of the 15-minute incubation, add ~3 ml of the NZY top agarose to the test tube containing the helper phage & E.coli. Remove the tube from the rack (which is still sitting in the water bath) and give it a quick flick of your wrist mixing the contents and immediately pour it onto an NZY plate. (I usually do three plates at one by pipetting 10ml of the NZY top agarose from the bottle, dispensing ~3.3 to one tube, ~3.3 to second tube and then another ~3.3 to a third tube. I then pour the plates starting with the first tube I put the NZY into.) Allow the top agarose to cool (~5 minutes). Invert the plate and incubate overnight at 37o C. Count the number of plaques. Determine the titer; i.e., pfu/ml using the formula: (Number of plaques (pfu) X Dilution Factor) X 1000ul/ml / Volume plated (µl)  Where the volume plated (in µl) refers to the volume of the helper phage solution added to the cells. Amplifying the Helper Phage Streak out E. coli strain XL1-Blue MRF′ onto an LBTet15 plate. Pick a single colony of XL1-Blue MRF′ and inoculate into 10ml 2X YT and grow until OD600 = 0.3 (~2.5 x 108 cells/ml). (No need to include the antibiotics in the overnight cultures.) Add helper phage at a multiplicity of infection (MOI) of 20:1 (phage-to-cells ratio). Do this for both ExAssist and VCSM13. Note: (If amplifying VCSM13 helper phage, add kanamycin to a final concentration of 25µg/ml to the medium 30 minutes after the helper phage and cells have been allowed to grow together.) Grow the culture at 37o C with vigorous aeration (~300RPM) for 8 hours. Heat the culture to 65o C for 15 minutes. Spin down the cell debris and transfer the supernatant to a fresh tube. Titer the helper phage produced. ExAssist should be 7.5 x 10¹⁰ to 1.0 x 10¹² pfu/ml and VCSM13 should be 1.0 x 10¹¹ to 1.0 x 10¹² pfu/ml Store at +4o C (Add DMSO to 7% for storage at –80oC). Notes: Several steps greatly aid the ease and success of pouring good phage onto plates: (1) Position the 37o C and 48o C water baths right next to each other with enough room in front of them for you to work. Wipe this area down with 70% EtOH prior to plating. (2) Use plates that have been prewarmed to 37oC (for one hour or more). (3) Use sterile glass pipettes that have been prewarmed to ~50o C. (4) Have a pipette holder in the work area. == Antibody Design Labs Phagemid Virion Production == A superinfection by a helper phage is necessary for phagemid pADL-22c-containing bacteria to produce virions. Please, consult the M13KO7 or CM13 helper phage manual for optimal conditions of superinfection. We recommend a rich medium such as 2xYT medium supplemented with ampicillin 100 µg/ml, kanamycin 50 µg/ml (when M13KO7 or CM13 helper phage are used), no glucose or less than 0.1% w/v, and incubation from 8 h to overnight at 30ºC and 250 rpm. Supplementation with IPTG is not necessary to achieve display on the phage with pADL-22c. We recommend adding the helper phage when the bacterial culture reaches an optical density at 600 nm between 0.4 OD and 0.5 OD; large amounts of non-superinfected cells due to immunity to superinfection will decrease virion production above 0.5 OD while disparities caused by differences pADL-22c Phagemid Instruction Manual 11 in phage growth rates will be amplified at a lower OD. Immunity to superinfection refers to the difficulty to transduce bacteria when protein III is expressed, as it is the case when with phagemids expressing a full-length pIII fusion protein. Notes Supplementation with IPTG is not necessary during virion production but is recommended for the expression of free antibodies in the periplasm with the non-amber suppressive SS320 bacterial strain.  Shorter incubation times 6 to 8 h long will produce less virions; we have not seen improvement of display on shorter incubation times; inversely, we have not seen sign of proteolysis of the linker after overnight incubation. Always use freshly prepared buffers from commercial concentrates during virion preparations to limit sources of proteolysis. Proteolysis usually occurs on concentrated virions; always prepare virions quickly and on ice. Kanamycin 50 µg/ml is enough to ensure selection with derivatives of M13KO7. Higher concentrations may be needed on very rich culture media or if your culture medium contains phosphate salts. [[Category:Antibodies]] [[Category:Protocols]]