Difference between revisions of "Team:Paris Bettencourt/Notebook/Protocols"
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| + | <div id=subheader> | ||
| + | <table> | ||
| + | <tr> | ||
| + | <td><a href="https://2016.igem.org/Team:Paris_Bettencourt/Notebook">Notebook</a></td> | ||
| + | <td><a href="#ancre">Protocols</a></td> | ||
| + | <td><a href="https://2016.igem.org/Team:Paris_Bettencourt/Notebook/Bibliography">Bibliography</a></td> | ||
| + | </tr> | ||
| + | </table> | ||
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| + | <div id="ancre"> | ||
| − | + | <h2 class="red">Phage Display Protocols</h2> | |
| − | + | ||
| − | + | <p> | |
| − | + | All protocols displayed here are identical to the ones provided in the NEB Ph.D.™-7 Phage Display Peptide Libraries Instruction Manual, except for the Panning Procedure that was adapted to our needs. | |
| − | + | </p> | |
| − | + | ||
| − | + | <h4>Phage Titering</h2> | |
| − | + | ||
| − | + | <p> | |
| − | + | The number of plaques will increase linearly with added phage only when the multiplicity of infection (MOI) is much less than 1 (i.e., cells are in considerable excess). For this reason, it is recommended that phage stocks be titered by diluting prior to infection, rather than by diluting cells infected at a high MOI. Plating at low MOI will also ensure that each plaque contains only one DNA sequence. | |
| − | + | </p> | |
| − | + | ||
| − | + | <ol> | |
| − | + | <li> | |
| − | + | Inoculate 10mL of LB with ER2738 from a plate and incubate with shaking ~3-4 hours (mid-log phase, OD600 ~ 0.5). | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | While cells are growing, melt Top Agar in microwave and dispense 3 ml into sterile culture tubes, one per expected phage dilution. Maintain tubes at 45°C. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Pre-warm, for at least one hour, one LB/IPTG/Xgal plate per expected dilu¬tion at 37°C until ready for use. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Prepare 10 to 103 -fold serial dilutions of phage in LB; 1 ml final volumes are convenient. Suggested dilution ranges: for amplified phage culture supernatants, 10^8 – 10^11; for unamplified panning eluates, 10^1–10^4. Use aerosol-resistant pipette tips to prevent cross-contamination, and use a fresh pipette tip for each dilution. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | When the culture in Step 1 reaches mid-log phase, dispense 200 μl into microfuge tubes, one for each phage dilution. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | To carry out infection, add 10 μl of each phage dilution to each tube, vortex quickly, and incubate at room temperature for 1–5 minutes. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Transfer the infected cells one infection at a time to culture tubes containing 45°C Top Agar. Vortex briefly and IMMEDIATELY pour culture onto a pre-warmed LB/IPTG/Xgal plate (200µM IPTG, 30µg/mL X-gal). Gently tilt and rotate plate to spread top agar evenly. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Allow the plates to cool for 5 minutes, invert, and incubate overnight at 37°C. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Count plaques on plates that have approximately 100 plaques. Multiply each number by the dilution factor for that plate to get phage titer in plaque forming units (pfu) per 10µL. Multiply by 100 to get the pfu/mL. | |
| − | + | </li> | |
| − | + | </ol> | |
| − | + | ||
| − | + | <h4>Panning Procedure</h4> | |
| − | + | ||
| − | + | <p> | |
| − | + | This protocol was adapted from the Surface Panning Procedure (Direct Target Coating) supplied in the NEB Ph.D.™-7 Phage Display Libraries Instruction Manual. | |
| − | + | </p> | |
| − | + | <h5>DAY 0</h5> | |
| − | + | ||
| − | + | <ul> | |
| − | + | <li> | |
| − | + | Streak ER2738 from glycerol stock on LB+Tet plate. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Incubate overnight at 4°C the piece of fabric/thread with 1mL of blocking buffer. Alternatively, after inoculation of the titering culture incubate the piece of fabric/thread at least 1 hour at 4°C on DAY 1. | |
| − | + | </li> | |
| − | + | </ul> | |
| − | + | ||
| − | + | <h5>DAY 1</h5> | |
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<ol> | <ol> | ||
<li> | <li> | ||
| − | Inoculate | + | Inoculate 10 ml of LB+Tet medium with ER2738 in a 125mL Erlenmeyer flask. This culture will be used for titering in Step 8 and can be used in ~3.5 - 4 hours. Incubate at 37°C with vigorous shaking. Incubate the titering culture until needed. |
| + | </li> | ||
| + | <li> | ||
| + | Remove the piece of fabric/thread from the blocking solution. Wash the fabric rapidly 10 times with TBST 0.1% (TBS + 0.1% [v/v] Tween-20) by dipping the threads in a well from a six-well plate filled with ~8mL TBST per well and going back and forth through the washing solution. Alternatively beackers can be used in place of six well plate. | ||
| + | </li> | ||
| + | <li> | ||
| + | Dilute a 25-fold representation of the library (e.g., 2 x 10 11 phage for a library with 2 x 10 9 clones) with 250µl of TBST (therefore add 10µL of phage library). Place the piece of fabric/thread in the Eppendorf tube containing the library and rock gently for 60 minutes at room temperature. | ||
</li> | </li> | ||
<li> | <li> | ||
| − | + | Inoculate 20mL of LB medium in a 250mL Erlenmeyer flask just before the end of the incubation period of the fabric in the phage library solution. Incubate at 37°C with vigorous shaking. This culture will be used to amplify the eluted phages. | |
</li> | </li> | ||
<li> | <li> | ||
| − | + | Remove piece of fabric/thread from the Eppendorf tube. | |
</li> | </li> | ||
<li> | <li> | ||
| − | + | Wash fabric 20 times with TBST as in step 2. | |
</li> | </li> | ||
<li> | <li> | ||
| − | + | Elute bound phage with 1 ml of an ap¬propriate elution buffer for the interaction being studied. A general buffer for nonspecific disruption of binding interactions is 0.2 M Glycine-HCl (pH 2.2), 1 mg/ml BSA. Rock gently for 15 minutes. Discard the piece of fabric. Neutralize supernatant with 150 μl of 1 M Tris-HCl, pH 9.1. | |
| + | </li> | ||
| + | <li> | ||
| + | Titer a small amount (~2 μl) of the eluate as described in the Phage Titering protocol. Plaques from the first or second round eluate titering can be sequenced if desired. | ||
| + | </li> | ||
| + | <li> | ||
| + | Amplify the rest of the eluate by adding the eluate to the 20-ml ER2738 culture from Step 4 (should be early-log at this point, i.e. OD600 ~ 0.01-0.05) and incubating with vigorous shaking for 4.5 hours at 37°C. | ||
| + | </li> | ||
| + | <em> | ||
| + | Note: The remaining eluate can be stored overnight at 4°C at this point, if preferred, and amplified the next day. In this case, inoculate 10 ml of LB+Tet with ER2738 and incubate with shaking overnight at 37°C. The next day, dilute the overnight culture 1:100 in 20 ml of LB in a 250-ml Erlenmeyer flask <strong>(do not use a 50 ml conical tube)</strong> and add the unamplified eluate. Incubate with vigorous shaking for 4.5 hours at 37°C and proceed to Step 10. | ||
| + | </em> | ||
| + | <li> | ||
| + | Transfer the culture to a centrifuge tube and spin for 10 minutes at 12,000 g at 4°C. Transfer the supernatant to a fresh tube and re-spin (discard the pellet). | ||
| + | </li> | ||
| + | <li> | ||
| + | Transfer the upper 80% of the supernatant to a fresh tube and add to it 1/6 volume of 20% PEG/2.5 M NaCl. Allow the phage to precipitate at 4°C for at least 2 hours, preferably overnight. | ||
</li> | </li> | ||
</ol> | </ol> | ||
| − | + | ||
| − | + | <h5>DAY 2</h5> | |
| − | + | ||
| − | + | <ol> | |
| − | + | <li> | |
| − | + | Inoculate 10mL of LB+Tet medium with ER2738 for titration. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Spin the PEG precipitation at 12,000 g for 15 minutes at 4°C. Decant and discard the supernatant, re-spin the tube briefly, and remove residual supernatant with a pipette. The phage pellet should be a white finger print sized smear on the side of the tube. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Suspend the pellet in 1 ml of TBS. Transfer the suspension to a micro¬centrifuge tube and spin at maximum (14,000 rpm) for 5 minutes at 4°C to pellet residual cells. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Transfer the supernatant to a fresh microcentrifuge tube and reprecipitate by adding 1/6 volume of 20% PEG/2.5 M NaCl. Incubate on ice for 15–60 minutes. Microcentrifuge at 14,000 rpm for 10 minutes at 4°C, discard the supernatant, re-spin briefly, and remove residual supernatant with a micropipet. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Suspend the pellet in 200 μl of TBS. Microcentrifuge for 1 minute to pel¬let any remaining insoluble material. Transfer the supernatant to a fresh tube. This is the amplified eluate. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Titer the amplified eluate as described in the Phage Titering protocol. The eluate can be stored for up to 3 weeks at 4°C. For long-term storage, add an equal volume of sterile glycerol and store at –20° C. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Incubate overnight at 4°C a new piece of fabric/thread in 1mL of blocking buffer for the 2nd round of panning. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Inoculate 10 ml of LB+Tet with ER2738 and incubate with shaking overnight at 37°C. | |
| − | + | </li> | |
| − | + | </ol> | |
| − | + | ||
| − | + | <h5>DAY 3</h5> | |
| − | + | ||
| − | + | <ol> | |
| − | + | <li> | |
| − | + | Count blue plaques from the titering plates in Step 6 of DAY 2 and determine the phage titer, which should be on the order of 1013–14 pfu/ml. Use this value to calculate an input volume corresponding to the input titer in Step 3 of DAY 1. If the phage titer of the amplified eluate is too low, succeeding rounds of panning can be carried out with as little as 109 pfu of input phage. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Carry out a second round of panning: repeat DAY 1 using the calculated amount of the first round amplified eluate as input phage, and raising the Tween concentration in the wash steps to 0.5% (v/v). | |
| − | + | </li> | |
| − | + | </ol> | |
| − | + | ||
| − | + | <h5>DAY 4</h5> | |
| − | + | ||
| − | + | <ol> | |
| − | + | <li> | |
| − | + | Repeat DAY 2 with the panning eluate from the 2nd round. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Incubate overnight at 4°C a new piece of fabric/thread with 1mL of blocking buffer for the 3rd round of panning. | |
| − | + | </li> | |
| − | + | </ol> | |
| − | + | ||
| − | + | <h5>DAY 5</h5> | |
| − | + | ||
| − | + | <ol> | |
| − | + | <li> | |
| − | + | Carry out a third round of panning: repeat DAY 1 (steps 1 to 9 only), using the second round amplified eluate at an input titer equivalent to what was used in the first round, again using 0.5% Tween in the wash steps. It is not necessary to amplify the third round eluate. <br> | |
| − | + | <em> | |
| − | + | Plaques from the titering can be used for sequencing: time the procedure so that plates are incubated at 37°C for no longer than 18 hours, as deletions may occur if plates are grown longer. Stored at 4°C, plates should be used to pick plaques within 1–3 days of plating. The remaining eluate can be stored at 4°C for at least one week. | |
| − | + | </em> | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | For preparation of individual clones for sequencing or ELISA, set up a 10-ml overnight culture of ER2738 from a colony, not by diluting the titering culture. Proceed with plaque amplification. Do not amplify the third round eluate and carry out a fourth round of panning unless the characterization shows no clear consensus sequence or phage ELISA results are negative. | |
| − | + | </li> | |
| − | + | </ol> | |
| − | + | ||
| − | + | <h4>Plaque Amplification for ELISA or Sequencing</h4> | |
| − | + | ||
| − | + | <p> | |
| − | + | Selected phage clones can be identified by DNA sequencing, and target specificity can be confirmed by phage ELISA. In both cases it is necessary to amplify phage, either from individual plaques or from the eluted pool, to obtain sufficient quantities to work with. | |
| − | + | </p> | |
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| − | + | <ol> | |
| − | + | <li> | |
| − | + | Dilute an overnight culture of ER2738 1:100 in LB. Dispense 1 ml of diluted culture into culture tubes, one for each clone to be characterized. 10–20 clones from the third round are usually sufficient to detect a consensus binding sequence. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Use a sterile wooden stick or pipette tip to stab a blue plaque from a titering plate (important: plates should be less than 1–3 days old, stored at 4°C and have less than 100 plaques) and transfer to a tube containing the diluted culture. Pick well-separated plaques. This will ensure that each plaque contains a single DNA sequence. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Incubate the tubes at 37°C with shaking for 4.5–5 hours (no longer). | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Transfer the cultures to microcentrifuge tubes, and microfuge at 14,000 rpm for 30 seconds (see next section to purify sequencing template). Transfer the supernatant to a fresh tube and re-spin. Using a pipette, transfer the upper 80% of the supernatant to a fresh tube. This is the amplified phage stock and can be stored at 4°C for several weeks with little loss of titer. For longterm storage (up to several years), dilute 1:1 with sterile glycerol and store at –20°C. | |
| − | + | </li> | |
| − | + | </ol> | |
| − | + | ||
| − | + | <h4>Sequencing of phage DNA</h4> | |
| − | + | ||
| − | + | <p> | |
| − | + | This extremely rapid procedure produces template of sufficient purity for manual or automated dideoxy sequencing, without the use of phenol or chromatography. | |
| − | + | </p> | |
| − | + | ||
| − | + | <ol> | |
| − | + | <li> | |
| − | + | Carry out the plaque amplification procedure described in the Phage Amplification protocol. After the first centrifugation in Step 4, transfer 500 µl of the phage-containing supernatant to a fresh microfuge tube. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Add 200 µl of 20% PEG/2.5 M NaCl. Invert several times to mix, and let stand for 10–20 minutes at room temperature. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Microfuge at 14,000 rpm for 10 minutes at 4°C and discard the supernatant. Phage pellet may not be visible. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Re-spin briefly. Carefully pipet away and discard any remaining supernatant. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Suspend the pellet thoroughly in 100 µl of Iodide Buffer by vigorously tapping the tube. Add 250 µl of ethanol and incubate 10–20 minutes at room temperature. Short incubation at room temperature will preferentially precipitate single-stranded phage DNA, leaving most phage protein in solution. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Spin in a microfuge at 14,000 rpm for 10 minutes at 4°C, and discard the supernatant. Wash the pellet with 0.5 ml of 70% ethanol (stored at –20°C), re-spin, discard the supernatant, and briefly dry the pellet under vacuum. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Suspend the pellet in 30 µl of TE buffer. The template can be suspended in H2O instead of TE if desired, but this is not recommended for long-term storage. In TE buffer, the phage DNA should be stable indefinitely at –20°C. | |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Quantitate product by using a Nanodrop. | |
| − | + | </li> | |
| − | + | </ol> | |
| − | + | ||
| − | + | <h5>Sequencing Guidelines</h5> | |
| − | + | ||
| − | + | <ol> | |
| − | + | <li> | |
| − | + | The sequence being read corresponds to the anticodon strand of the template. Write out the complementary strand and check against the top strand of the insert sequence shown in the figure below (the primers hybridize downstream of the insert). Check that the third nucleotide of each codon in the randomized region is G or T. | |
| − | + | </li> | |
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| − | The | + | |
| − | </ | + | |
| − | + | ||
<div style="text-align:center;"> | <div style="text-align:center;"> | ||
<img src="https://static.igem.org/mediawiki/2016/d/dd/Paris_Bettencourt-Sequencing_scheme.jpg" alt="Sequencing_scheme_NEB_PhD"/> | <img src="https://static.igem.org/mediawiki/2016/d/dd/Paris_Bettencourt-Sequencing_scheme.jpg" alt="Sequencing_scheme_NEB_PhD"/> | ||
</div> | </div> | ||
| − | + | <li> | |
| − | < | + | TAG stop codons are suppressed by glutamine in ER2738 (glnV). If the library was amplified in this strain or any glnV (also known as supE) strain, TAG should be considered a glutamine codon when translating. |
| − | + | </li> | |
| − | + | <li> | |
| − | + | Libraries often contain a small percentage (less than 1%) of clones containing multiple inserts of the randomized region. Preferential selection and amplification of these clones may occur when panning against targets whose ligand specificity spans a length greater than that specified by the insert. When interpreting sequence data, be sure the sequence outside the restriction sites used for inserting the randomized sequence (Acc65I and EagI) matches the sequence shown in Figure 5. Once the flanking regions of the library insert have been identified, the insert size is easily determined. | |
| − | + | </li> | |
| − | </ | + | </ol> |
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| − | + | </div> | |
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Revision as of 18:13, 28 August 2016
Phage Display Protocols
All protocols displayed here are identical to the ones provided in the NEB Ph.D.™-7 Phage Display Peptide Libraries Instruction Manual, except for the Panning Procedure that was adapted to our needs.
Phage Titering
The number of plaques will increase linearly with added phage only when the multiplicity of infection (MOI) is much less than 1 (i.e., cells are in considerable excess). For this reason, it is recommended that phage stocks be titered by diluting prior to infection, rather than by diluting cells infected at a high MOI. Plating at low MOI will also ensure that each plaque contains only one DNA sequence.
- Inoculate 10mL of LB with ER2738 from a plate and incubate with shaking ~3-4 hours (mid-log phase, OD600 ~ 0.5).
- While cells are growing, melt Top Agar in microwave and dispense 3 ml into sterile culture tubes, one per expected phage dilution. Maintain tubes at 45°C.
- Pre-warm, for at least one hour, one LB/IPTG/Xgal plate per expected dilu¬tion at 37°C until ready for use.
- Prepare 10 to 103 -fold serial dilutions of phage in LB; 1 ml final volumes are convenient. Suggested dilution ranges: for amplified phage culture supernatants, 10^8 – 10^11; for unamplified panning eluates, 10^1–10^4. Use aerosol-resistant pipette tips to prevent cross-contamination, and use a fresh pipette tip for each dilution.
- When the culture in Step 1 reaches mid-log phase, dispense 200 μl into microfuge tubes, one for each phage dilution.
- To carry out infection, add 10 μl of each phage dilution to each tube, vortex quickly, and incubate at room temperature for 1–5 minutes.
- Transfer the infected cells one infection at a time to culture tubes containing 45°C Top Agar. Vortex briefly and IMMEDIATELY pour culture onto a pre-warmed LB/IPTG/Xgal plate (200µM IPTG, 30µg/mL X-gal). Gently tilt and rotate plate to spread top agar evenly.
- Allow the plates to cool for 5 minutes, invert, and incubate overnight at 37°C.
- Count plaques on plates that have approximately 100 plaques. Multiply each number by the dilution factor for that plate to get phage titer in plaque forming units (pfu) per 10µL. Multiply by 100 to get the pfu/mL.
Panning Procedure
This protocol was adapted from the Surface Panning Procedure (Direct Target Coating) supplied in the NEB Ph.D.™-7 Phage Display Libraries Instruction Manual.
DAY 0
- Streak ER2738 from glycerol stock on LB+Tet plate.
- Incubate overnight at 4°C the piece of fabric/thread with 1mL of blocking buffer. Alternatively, after inoculation of the titering culture incubate the piece of fabric/thread at least 1 hour at 4°C on DAY 1.
DAY 1
- Inoculate 10 ml of LB+Tet medium with ER2738 in a 125mL Erlenmeyer flask. This culture will be used for titering in Step 8 and can be used in ~3.5 - 4 hours. Incubate at 37°C with vigorous shaking. Incubate the titering culture until needed.
- Remove the piece of fabric/thread from the blocking solution. Wash the fabric rapidly 10 times with TBST 0.1% (TBS + 0.1% [v/v] Tween-20) by dipping the threads in a well from a six-well plate filled with ~8mL TBST per well and going back and forth through the washing solution. Alternatively beackers can be used in place of six well plate.
- Dilute a 25-fold representation of the library (e.g., 2 x 10 11 phage for a library with 2 x 10 9 clones) with 250µl of TBST (therefore add 10µL of phage library). Place the piece of fabric/thread in the Eppendorf tube containing the library and rock gently for 60 minutes at room temperature.
- Inoculate 20mL of LB medium in a 250mL Erlenmeyer flask just before the end of the incubation period of the fabric in the phage library solution. Incubate at 37°C with vigorous shaking. This culture will be used to amplify the eluted phages.
- Remove piece of fabric/thread from the Eppendorf tube.
- Wash fabric 20 times with TBST as in step 2.
- Elute bound phage with 1 ml of an ap¬propriate elution buffer for the interaction being studied. A general buffer for nonspecific disruption of binding interactions is 0.2 M Glycine-HCl (pH 2.2), 1 mg/ml BSA. Rock gently for 15 minutes. Discard the piece of fabric. Neutralize supernatant with 150 μl of 1 M Tris-HCl, pH 9.1.
- Titer a small amount (~2 μl) of the eluate as described in the Phage Titering protocol. Plaques from the first or second round eluate titering can be sequenced if desired.
- Amplify the rest of the eluate by adding the eluate to the 20-ml ER2738 culture from Step 4 (should be early-log at this point, i.e. OD600 ~ 0.01-0.05) and incubating with vigorous shaking for 4.5 hours at 37°C. Note: The remaining eluate can be stored overnight at 4°C at this point, if preferred, and amplified the next day. In this case, inoculate 10 ml of LB+Tet with ER2738 and incubate with shaking overnight at 37°C. The next day, dilute the overnight culture 1:100 in 20 ml of LB in a 250-ml Erlenmeyer flask (do not use a 50 ml conical tube) and add the unamplified eluate. Incubate with vigorous shaking for 4.5 hours at 37°C and proceed to Step 10.
- Transfer the culture to a centrifuge tube and spin for 10 minutes at 12,000 g at 4°C. Transfer the supernatant to a fresh tube and re-spin (discard the pellet).
- Transfer the upper 80% of the supernatant to a fresh tube and add to it 1/6 volume of 20% PEG/2.5 M NaCl. Allow the phage to precipitate at 4°C for at least 2 hours, preferably overnight.
DAY 2
- Inoculate 10mL of LB+Tet medium with ER2738 for titration.
- Spin the PEG precipitation at 12,000 g for 15 minutes at 4°C. Decant and discard the supernatant, re-spin the tube briefly, and remove residual supernatant with a pipette. The phage pellet should be a white finger print sized smear on the side of the tube.
- Suspend the pellet in 1 ml of TBS. Transfer the suspension to a micro¬centrifuge tube and spin at maximum (14,000 rpm) for 5 minutes at 4°C to pellet residual cells.
- Transfer the supernatant to a fresh microcentrifuge tube and reprecipitate by adding 1/6 volume of 20% PEG/2.5 M NaCl. Incubate on ice for 15–60 minutes. Microcentrifuge at 14,000 rpm for 10 minutes at 4°C, discard the supernatant, re-spin briefly, and remove residual supernatant with a micropipet.
- Suspend the pellet in 200 μl of TBS. Microcentrifuge for 1 minute to pel¬let any remaining insoluble material. Transfer the supernatant to a fresh tube. This is the amplified eluate.
- Titer the amplified eluate as described in the Phage Titering protocol. The eluate can be stored for up to 3 weeks at 4°C. For long-term storage, add an equal volume of sterile glycerol and store at –20° C.
- Incubate overnight at 4°C a new piece of fabric/thread in 1mL of blocking buffer for the 2nd round of panning.
- Inoculate 10 ml of LB+Tet with ER2738 and incubate with shaking overnight at 37°C.
DAY 3
- Count blue plaques from the titering plates in Step 6 of DAY 2 and determine the phage titer, which should be on the order of 1013–14 pfu/ml. Use this value to calculate an input volume corresponding to the input titer in Step 3 of DAY 1. If the phage titer of the amplified eluate is too low, succeeding rounds of panning can be carried out with as little as 109 pfu of input phage.
- Carry out a second round of panning: repeat DAY 1 using the calculated amount of the first round amplified eluate as input phage, and raising the Tween concentration in the wash steps to 0.5% (v/v).
DAY 4
- Repeat DAY 2 with the panning eluate from the 2nd round.
- Incubate overnight at 4°C a new piece of fabric/thread with 1mL of blocking buffer for the 3rd round of panning.
DAY 5
-
Carry out a third round of panning: repeat DAY 1 (steps 1 to 9 only), using the second round amplified eluate at an input titer equivalent to what was used in the first round, again using 0.5% Tween in the wash steps. It is not necessary to amplify the third round eluate.
Plaques from the titering can be used for sequencing: time the procedure so that plates are incubated at 37°C for no longer than 18 hours, as deletions may occur if plates are grown longer. Stored at 4°C, plates should be used to pick plaques within 1–3 days of plating. The remaining eluate can be stored at 4°C for at least one week. - For preparation of individual clones for sequencing or ELISA, set up a 10-ml overnight culture of ER2738 from a colony, not by diluting the titering culture. Proceed with plaque amplification. Do not amplify the third round eluate and carry out a fourth round of panning unless the characterization shows no clear consensus sequence or phage ELISA results are negative.
Plaque Amplification for ELISA or Sequencing
Selected phage clones can be identified by DNA sequencing, and target specificity can be confirmed by phage ELISA. In both cases it is necessary to amplify phage, either from individual plaques or from the eluted pool, to obtain sufficient quantities to work with.
- Dilute an overnight culture of ER2738 1:100 in LB. Dispense 1 ml of diluted culture into culture tubes, one for each clone to be characterized. 10–20 clones from the third round are usually sufficient to detect a consensus binding sequence.
- Use a sterile wooden stick or pipette tip to stab a blue plaque from a titering plate (important: plates should be less than 1–3 days old, stored at 4°C and have less than 100 plaques) and transfer to a tube containing the diluted culture. Pick well-separated plaques. This will ensure that each plaque contains a single DNA sequence.
- Incubate the tubes at 37°C with shaking for 4.5–5 hours (no longer).
- Transfer the cultures to microcentrifuge tubes, and microfuge at 14,000 rpm for 30 seconds (see next section to purify sequencing template). Transfer the supernatant to a fresh tube and re-spin. Using a pipette, transfer the upper 80% of the supernatant to a fresh tube. This is the amplified phage stock and can be stored at 4°C for several weeks with little loss of titer. For longterm storage (up to several years), dilute 1:1 with sterile glycerol and store at –20°C.
Sequencing of phage DNA
This extremely rapid procedure produces template of sufficient purity for manual or automated dideoxy sequencing, without the use of phenol or chromatography.
- Carry out the plaque amplification procedure described in the Phage Amplification protocol. After the first centrifugation in Step 4, transfer 500 µl of the phage-containing supernatant to a fresh microfuge tube.
- Add 200 µl of 20% PEG/2.5 M NaCl. Invert several times to mix, and let stand for 10–20 minutes at room temperature.
- Microfuge at 14,000 rpm for 10 minutes at 4°C and discard the supernatant. Phage pellet may not be visible.
- Re-spin briefly. Carefully pipet away and discard any remaining supernatant.
- Suspend the pellet thoroughly in 100 µl of Iodide Buffer by vigorously tapping the tube. Add 250 µl of ethanol and incubate 10–20 minutes at room temperature. Short incubation at room temperature will preferentially precipitate single-stranded phage DNA, leaving most phage protein in solution.
- Spin in a microfuge at 14,000 rpm for 10 minutes at 4°C, and discard the supernatant. Wash the pellet with 0.5 ml of 70% ethanol (stored at –20°C), re-spin, discard the supernatant, and briefly dry the pellet under vacuum.
- Suspend the pellet in 30 µl of TE buffer. The template can be suspended in H2O instead of TE if desired, but this is not recommended for long-term storage. In TE buffer, the phage DNA should be stable indefinitely at –20°C.
- Quantitate product by using a Nanodrop.
Sequencing Guidelines
- The sequence being read corresponds to the anticodon strand of the template. Write out the complementary strand and check against the top strand of the insert sequence shown in the figure below (the primers hybridize downstream of the insert). Check that the third nucleotide of each codon in the randomized region is G or T.
- TAG stop codons are suppressed by glutamine in ER2738 (glnV). If the library was amplified in this strain or any glnV (also known as supE) strain, TAG should be considered a glutamine codon when translating.
- Libraries often contain a small percentage (less than 1%) of clones containing multiple inserts of the randomized region. Preferential selection and amplification of these clones may occur when panning against targets whose ligand specificity spans a length greater than that specified by the insert. When interpreting sequence data, be sure the sequence outside the restriction sites used for inserting the randomized sequence (Acc65I and EagI) matches the sequence shown in Figure 5. Once the flanking regions of the library insert have been identified, the insert size is easily determined.
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24, rue du Faubourg Saint Jacques
75014 Paris, France
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igem2016parisbettencourt@gmail.com
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+33 1 44 41 25 22/25
igem2016parisbettencourt@gmail.com 