Team:Paris Bettencourt/Notebook/Binding


Week 27th June - 3rd July

Week 11th - 17th July

NEB Ph.D.™-7 Phage Display Peptide Library Kit solutions preparation

NEB protocol recommends to prepare a mixed solution of ITPG and X-gal. Because I already have IPTG stock, I prefer to do 2 separate solution for IPTG and X-gal (also avoids throwing both IPTG and X-gal if anything were to happen to the tubes).

IPTG

M.W.= 238.31g/mol
NEB protocol recommends to put 1.25g in 25mL DMF, which is [IPTG]=50mg/mL
[IPTG]mass=[IPTG]/MW=0.05/238.31=0.21mol/L=210mM.
They recommend diluting 1000-fold to prepare plates. So [IPTG]working_concentration=50µg/mL which is 210µM.
In order to simplify things, I'll use [IPTG]working_concentration=200µM.
IPTG stocks are at 1M and 0.1M (100mM).
If preparing 1M stock, add 2.383g of IPTG to 10mL of milli-Q water. For 0.1M, add 238.3mg of IPTG to 10mL of milli-Q water.
2µL IPTG / mL medium should be added if using the 0.1M solution (0.2µL / mL of using the 1M solution).

X-gal

M.W.= 408.63g/mol
NEB protocol recommends to put 1g in 25mL DMF, which is [X-gal]=40mg/mL.
Sigma-Aldrich recommends having stock solution at 20mg/mL.
I decided to do a stock at 20mg/mL.
2 µL X-gal / mL of medium should be added.

Tetracycline

NEB protocol recommends doing a 20mg/mL stock solution in 50% EtOH. Most of the protocol found online recommend 70% EtOH, so that's what I did (it probably does not matter much).
300mg were dissolved in 15mL 70% EtOH.

TBS

NEB protocol recommends 50mM Tris-HCl (pH7.5), 150mM NaCl.

Tris-HCl 1M, pH 7.5:
M.W. Tris-Base=121.14g/mol
12.11g of Tris-Base were dissolved in 60mL of H2O.
pH was adjusted to 7.5 using HCl 5M.
Volume was adjusted to 100mL.

25 mL of Tris-HCl 1M, pH 7.5 were diluted in 500mL H2O.
M.W. NaCl=58.44g/mol
mNaCl=(58.44/1000)*150=8.766g for 1L
So 8.766/2=4.383g were added to the solution of Tris-HCl 50mM.
pH was measured after completion of the solution and was around 7.7.

Blocking Buffer

M.W. NaHCO3=84.007g/mol
2.1g of NaHCO3 were put in 225mL osmosed H2O.
pH was adjusted to 8.6 with 5M HCl.
Volume was adjusted to 250mL.
1.25 BSA was added to final concentration 5mg/mL.
Solution was kept 6 days at 4°C.
On 21/07/16, 0.05g NaN3 (0.02%) were added to the solution to avoid growth of microorganisms.
Solution was filter sterilized and aliquoted by ~75mL.

Week 18th - 24th July

M13KE Phage Titering

In order to train ourselves with the phage titration that we will need to perform when doing the phage display from the NEB kit, we decided to do a phage titration using M13KE. The following protocol is the one given by NEB with their M13KE phages.

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).

Note: We used MGZ1 F+ to perform this titration

  1. Inoculate 10 ml of LB with both MGZ1 F+ from a plate and incubate with shak­ing 4–6 hrs (mid-log phase, OD600 ~ 0.5).
    Media was inoculated at 8h56.
    Time after inoculation OD 600nm
    3h14 0.0584
    4h11 0.2717
    4h28 0.4022
    4h28 0.5644
  2. 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.
  3. Pre-warm, for at least one hour, one LB/IPTG/Xgal plate per expected dilu­tion at 37°C until ready for use.
  4. Prepare 10 to 1000-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.
    As I expect to have 10^11 pfu/mL after amplification and that they recommend 10^8 to 10^11 dilutions from that, beginning from M13KE tube (10^13 pfu/mL), I used the following dilutions: 10^10, 10^11, 10^12 and 10^13 (shift of 2 order of magnitude).
  5. When the culture in Step 1 reaches mid-log phase, dispense 200 μl into microfuge tubes, one for each phage dilution.
  6. 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. Here cells were incubated with the phages 3 to 4 minutes.
  7. 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. Gently tilt and rotate plate to spread top agar evenly.
  8. Allow the plates to cool for 5 minutes, invert, and incubate overnight at 37°C.
  9. 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 10 μl.

Results:

Dilution Number of plaques
10^10 25
10^11 16
10^12 26
10^13 16

M13KE phage amplification

This phage amplification protocol is the one given by NEB.

  1. 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, 150 rpm for 4 -5 hrs.
  2. Remove cells by centrifugation at 4500 g for 10 min. Transfer supernatant to a fresh tube. Repeat centrifugation.
  3. 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.
  4. Pellet phage by centrifugation at 12000 g for 15 min. 55 min at 4000 rpm (Rotor radius: 195 mm, 4000 rpm -> ~3400 g, 12000/~3400=3.5, 3.5*15 min=52.5 min) used was done instead due to problems with centrifuge. Decant supernatant. Resuspend pellet in 1 mL TBS. Transfer to an eppendorf tube. Spin briefly to remove any cell debris.
  5. Transfer supernatant to a fresh tube. Add 200 µL of 2.5 M NaCl/20% PEG-8000. Incubate on ice for 60 min. Spin 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.

Week 25th - 31th July

NEB Phage Display protocol on cotton - DAY 1

Cotton used: Khadi (from Khadi and Co - Bess Nielsen, given by Teja) - Hand Woven - 100% cotton.
A square of 1cm x 1cm was used (total surface: 2 cm² counting both sides).

  1. Inoculate 10 ml of LB+Tet medium with ER2738. This culture will be used for titering in Step 8 and can be used in ~4 hours. Incubate at 37°C with vigorous shaking. Incubate the titering culture until needed.
  2. Incubate for at least 1 hour at 4°C a piece of fabric with blocking buffer.
  3. Remove the piece of fabric from the blocking solution. Wash the fabric rapidly 10 times with TBST (TBS + 0.1% [v/v] Tween-20) by dipping the fabric in a beacker filled with TBST and going back and forth through the washing solution.
  4. 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). Pipette onto piece of fabric in Eppendorf tube and rock gently for 60 minutes at room temperature.
    Rem: The square shape of the piece of fabric does not seem best suited for the incubaction with the phages as the sides seem not to be immersed in the phage library solution. It is probably better to use a rectangle shape thereafter so that the piece of fabric fits nicely into the tube and is fully immersed.
  5. Remove piece of fabric from the Eppendorf tube.
  6. Wash fabric 20 times with TBST as in step 3.
  7. Elute bound phage with 1 ml of an ap­propriate 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 (save it in TBST). Neutralize supernatant with 150 μl of 1 M Tris-HCl, pH 9.1.
  8. Inoculate 20 mL of LB medium in a 250-ml Erlenmeyer flask with ER2738. Incubate culture at 37°C with vigorous shaking. Carefully, monitor the 20-ml culture so that it does not grow beyond early-log phase (OD600 0.01–0.05), for use in Step 10.
  9. Titer a small amount (~2 μl) of the eluate as described in General M13 titration protocol. Plaques from the first or second round eluate titering can be sequenced if desired.
    Rem: As this eluate is not amplified, dilutions used for titration are as follow: 10, 100, 1000, 10000.
  10. Amplify the rest of the eluate by adding the eluate to the 20-ml ER2738 culture from Step 1 (should be early-log at this point) and incubating with vigorous shaking for 4.5 hours at 37°C.
  11. 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).
  12. Transfer the upper 80% (here 12.8mL) of the supernatant to a fresh tube and add to it 1/6 volume of 20% PEG/2.5 M NaCl (here ~2.1mL). Allow the phage to precipitate at 4°C for at least 2 hours, preferably overnight.
Time after inoculation - Titration culture OD 600nm - Titration culture Time after inoculation - Amplification culture OD 600nm - 20mL Amplification culture
0 0 0 0
3h30 0.3811 0h40 0.008/0.006/0.009
3h40 0.5101 1h50 0.0116
2h25 0.0193

NEB Phage Display protocol on cotton - DAY 2

Results titration unamplified phage eluate 1st round:

Dilution Number of plaques
10 >1000
100 230
1000 17
10000 3

Therefore, we have a phage concentration of 230x(10^2)x(10^2) = 2.3 x 10^6 pfu/mL.

  1. Inoculate 10mL of LB+Tet medium with ER2738 for titration.
  2. 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.
  3. Suspend the pellet in 1 ml of TBS. Transfer the suspension to a micro­centrifuge tube and spin at maximum (14,000 rpm) for 5 minutes at 4°C to pellet residual cells.
  4. Transfer the supernatant to a fresh microcentrifuge tube and reprecipitate by adding 1/6 volume of 20% PEG/2.5 M NaCl (here 166µL). 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.
  5. Suspend the pellet in 200 μl of TBS. Microcentrifuge for 1 minute to pel­let any remaining insoluble material. Transfer the supernatant to a fresh tube. This is the amplified eluate.
  6. Titer the amplified eluate on LB/IPTG/Xgal plates following the general M13 titration 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.
    OD measurements:
    Time after inoculation OD 600nm
    0 0
    3h08 0.3167
    3h21 0.4415
    3h25 0.4955
  7. Incubate overnight at 4°C a new piece of fabric (both a 1 cm x 1cm square and 0.5 cm x 2 cm were incubated) in 1 mL of Blocking Buffer for the 2 nd round of panning.

    8. Week 01st - 07 August

    NEB Phage Display protocol on cotton - DAY 3:

    1. Count blue plaques from the titering plates in Step 17 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 4. 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.
      Results titration amplified phage eluate 1st round:
      Dilution Number of plaques
      10^8 >1000
      10^9 92
      10^10 8
      10^11 1
      Therefore, we have a phage concentration of 92x(10^9)x(10^2) = 9.2 x 10^12 pfu/mL.
    2. Inoculate 10 ml of LB+Tet medium with ER2738. This culture will be used for titering in Step 9 and can be used in ~4 hours. Incubate at 37°C with vigorous shaking. Incubate the titering culture until needed.
    3. Remove the piece of fabric from the blocking solution. Wash the fabric rapidly 10 times with TBST (TBS + 0.5% [v/v] Tween-20) by dipping the fabric in a beacker filled with TBST and going back and forth through the washing solution.
    4. Dilute a 11.5-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). Pipette onto piece of fabric in Eppendorf tube and rock gently for 60 minutes at room temperature.
      Rem: The square shape of the piece of fabric does not seem best suited for the incubaction with the phages as the sides seem not to be immersed in the phage library solution. It is probably better to use a rectangle shape thereafter so that the piece of fabric fits nicely into the tube and is fully immersed.
    5. Remove piece of fabric from the Eppendorf tube.
    6. Wash fabric 20 times with TBST as in step 3.
    7. Elute bound phage with 1 ml of an ap­propriate 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 (save it in TBST). Neutralize supernatant with 150 μl of 1 M Tris-HCl, pH 9.1.
    8. Inoculate 20 mL of LB medium in a 250-ml Erlenmeyer flask with ER2738. Incubate culture at 37°C with vigorous shaking. Carefully, monitor the 20-ml culture so that it does not grow beyond early-log phase (OD600 0.01–0.05), for use in Step 10.
    9. Titer a small amount (~2 μl) of the eluate on LB/IPTG/Xgal following general M13 titration protocol. Plaques from the first or second round eluate titering can be sequenced if desired.
      OD measuerments:
      Time after inoculation OD 600nm
      0 0
      3h33 0.1130
      4h03 0.2107
      4h35 0.3251
      4h50 0.5356
    10. Amplify the rest of the eluate by adding the eluate to the 20-ml ER2738 culture from Step 1 (should be early-log at this point) and incubating with vigorous shaking for 4.5 hours at 37°C.
      OD measuerments:
      Time after inoculation OD 600nm
      0 0
      1h01 0
      1h33 0.006
      2h06 0.0388
    11. 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).
    12. Transfer the upper 80% (here 14.4mL) of the supernatant to a fresh tube and add to it 1/6 volume of 20% PEG/2.5 M NaCl (here 2.4mL). Allow the phage to precipitate at 4°C for at least 2 hours, preferably overnight.

    NEB Phage Display protocol on cotton - DAY 4

    Results titration unamplified phage eluate 2nd round:

    Dilution Number of plaques
    10 >1000
    100 788
    1000 38
    10000 10

    Therefore, we have a phage concentration of 38x(10^3)x(10^2) = 3.8 x 10^6 pfu/mL

    1. Inoculate 10mL of LB+Tet medium with ER2738 for titration.
    2. 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.
    3. Suspend the pellet in 1 ml of TBS. Transfer the suspension to a micro­centrifuge tube and spin at maximum (14,000 rpm) for 5 minutes at 4°C to pellet residual cells.
    4. Transfer the supernatant to a fresh microcentrifuge tube and reprecipitate by adding 1/6 volume of 20% PEG/2.5 M NaCl (here 166µL). 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.
    5. Suspend the pellet in 200 μl of TBS. Microcentrifuge for 1 minute to pel­let any remaining insoluble material. Transfer the supernatant to a fresh tube. This is the amplified eluate.
    6. Titer the amplified eluate on LB/IPTG/Xgal plates following the general M13 titration 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.
      OD measurements:
      Time after inoculation OD 600nm
      0 0
      3h38 1.032 => 10-fold dilution
      4h18 0.2009
      4h42 0.42
      4h48 0.5094

    NEB Phage Display protocol on cotton - DAY 5

    1. Count blue plaques from the titering plates in Step 17 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 4. 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.
      Results titration amplified phage eluate 1st round:
      Dilution Number of plaques
      10^8 >1000
      10^9 277
      10^10 40
      10^11 4
      Therefore, we have a phage concentration of (277x(10^9)x(10^2)+40x(10^10)x(10^2))/2 = 3.36 x 10^13 pfu/mL
    2. Inoculate 10 ml of LB+Tet medium with ER2738. This culture will be used for titering in Step 8 and can be used in ~4 hours. Incubate at 37°C with vigorous shaking. Incubate the titering culture until needed.
    3. Remove the piece of fabric from the blocking solution. Wash the fabric rapidly 10 times with TBST (TBS + 0.5% [v/v] Tween-20) by dipping the fabric in a beacker filled with TBST and going back and forth through the washing solution.
    4. Dilute a 168-fold representation of the library with 250µl of TBST (therefore add 1.5µL of phage library to 248.5µL TBST). Pipette onto piece of fabric in Eppendorf tube and rock gently for 60 minutes at room temperature.
    5. Remove piece of fabric from the Eppendorf tube.
    6. Wash fabric 20 times with TBST as in step 3.
    7. Elute bound phage with 1 ml of an ap­propriate 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 (save it in TBST). Neutralize supernatant with 150 μl of 1 M Tris-HCl, pH 9.1.
    8. Titer a small amount (~2 μl) of the eluate on LB/IPTG/Xgal following general M13 titration protocol. Plaques from the first or second round eluate titering can be sequenced if desired.
      OD measuerments:
      Time after inoculation OD 600nm
      0 0
      3h16 0.2936
      3h45 0.2936
      4h02 0.4242
      4h11 0.5237
    9. It is not necessary to amplify the third round eluate. Plaques from this 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.

    Plaque amplification Sequencing - Cotton

    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.

    1. 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. 20 clones were selected.
    2. Use a sterile wooden stick or pipette tip (tip used here) to stab a blue plaque from a titering plate (important: plates should be <1–3 days old, stored at 4°C and have <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.
    3. Incubate the tubes at 37°C with shaking for 4.5–5 hours (4h45').
    4. Transfer the cultures to microcentrifuge tubes, and microfuge at 14,000 rpm for 30 seconds. 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 long-term storage (up to several years), dilute 1:1 with sterile glycerol and store at –20°C.

    Week 08th - 14th August

    Sequencing of Phage DNA from Cotton Phage Display

    1. After the first centrifugation in Step 4 in the phage amplification protocol, transfer 500 μl of the phage-containing supernatant to a fresh microfuge tube.
    2. 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.
      3. Microfuge at 14,000 rpm for 10 minutes at 4°C and discard the supernatant. Phage pellet may not be visible.
    3. Re-spin briefly. Carefully pipet away and discard any remaining supernatant.
      4. 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.
    4. 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 dry pellet (here it was dried 1h at RT with ~15µL EtOH remaining after discarding supernatant).
    5. Suspend the pellet in 30 μl of H2O. The template can be suspended in TE if desired, this is recommended for long-term storage. In TE buffer, the phage DNA should be stable indefinitely at –20°C.
    6. Quantitate product by using a Nanodrop.
      Note: Make sure to use single strand Dna measurement
      Sample Concentration (ng/µL) 260/280 ration
      R3P1 52 ND
      R3P2 57 ND
      R3P3 14 ND
      R3P4 68 2.46
      R3P5 29 2.53
      R3P6 61 2.00
      R3P7 97 2.01
      R3P8 61 1.97
      R3P9 61 2.55
      R3P10 41 2.16
      R3P11 80 2.04
      R3P12 64 2.04
      R3P13 64 2.15
      R3P14 66 2.02
      R3P15 65 1.96
      R3P16 44.5 2.05
      R3P17 45 2.10
      R3P18 50 2.00
      R3P19 43 2.00
      R3P20 53 1.90

    All samples were sent to sequencing by GATC Biotech.
    Results are available below:

    Sequences Phage R3P6 to R3P15
    Sequences Phage R3P1 to R3P5 and R3P16 to R3P20

    NEB Phage Display on Cotton - Finding a consensus sequence

    Sequences were analysed using Geneious 9.1.5.
    The random peptide library sequence was found between the linker (motif: 5'-ACC TCC ACC-3') and the end of the pIII leader sequence (motif: 5'-AGA GTG AGA-3'). We used only the -96 primer (see sequence below) to sequence as the -28 seemed too close to the library sequence for the sequencing to give a good result.

    Sequencing_scheme_NEB_PhD

    Some samples (R3P4, R3P13, R3P17) did not exhibit those motifs. Deletion?
    One of the sequences was longer (22 nucleotides) and contained non-determined nucleotides (N).
    Those four sequences were not used in the analysis.
    The 21 nucleotides of the library of 16 remaining sequences were then translated.

    Translation_sequence_random_peptide_library_NEB_PhD_Cotton

    Using the Multiple-Align function of Geneious and setting the threshold for the consensus sequence at 25% (amino acid present at least 25% of the time at a given position), we get the following consensus sequence:

    Consensus_sequence_random_peptide_library_NEB_PhD_Cotton

    Week 15th - 21st August

    Week 22nd - 28th August

    Week 29th August - 4th September

    Week 5th September - 11th September


Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
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+33 1 44 41 25 22/25
igem2016parisbettencourt@gmail.com
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