Difference between revisions of "Team:Paris Bettencourt/Notebook/Binding"

Introduction

Binding domain group aims at finding novel peptides that can bind tightly and specifically to a particular fabric. We hope to improve the efficiency of enzymes tested by the Enzymes search group by localizing and stabilizing the enzymes at the sites of action (fabric) by fusing the peptide sequences we find to those enzymes. To achieve this goal we are using phage display technique.

Phage display describes a selection technique in which phages with genetic material encoding variants of peptide sequences express these peptides on the protein coat. And based on the binding affinity of these peptides to a given target molecule by an in vitro selection process called panning, selective phages can be separated and enriched. We adapted the NEB protocol to accommodate our target, so panning is carried out by incubating a library of phage-displayed peptides (2*10^11 phages) with our fabric of choice in a micro centrifuge tube, washing away the unbound phage, and eluting the specifically bound phage. The eluted phage are then amplified and taken through additional binding/amplification cycles to enrich the pool in favor of binding se¬quences. After 3–4 rounds of panning, individual clones will be characterized by DNA sequenc¬ing and later ELISA.

The peptide library we received from NEB has 10^9 distinct clones. The M13 phages are modified for a pentavalent display of peptides as N-terminal fusions to the minor coat protein pIII. This protein modulates phage infectivity by binding to the F-pilus of the recipient bacterial cell. The E. coli host strain used is ER2738 (F´ proA+B+ lacIq Δ(lacZ)M15 zzf::Tn10(TetR)/fhuA2 glnV Δ(lac-proAB) thi-1 Δ(hsdS-mcrB)5. [rk– mk– McrBC–]), which is a robust F+ strain with a rapid growth rate and is supE (GlnV) in order to suppress amber (UAG) stop codons within the library with glutamine.

In summary, the main focus of our group is to find novel peptide sequences that specifically bind to different fabrics. Later, collaborate with other groups to optimize the efficiency of the potential enzymes they find on wine stained fabrics using our peptide sequences.

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 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 beaker 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 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.

Week 01st - 07 August

NEB Phage Display 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 beaker 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 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 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 beaker 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

Purification of Phage DNA from Cotton Phage Display for sequencing

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.
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.
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 ratio
   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

Sequencing of phage DNA from NEB phage Display on Cotton

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.



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.



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:



NEB Phage Display on Polyester and Silk - DAY 1 (Part 1)

Silk used: Silk thread 1003 Au ver a Soie - Colour: Crème (Cream) - 100% Soie - Length: 50 cm - Weight: 16.5mg.
Polyester used: Polyester thread Mediac Ref: 960 - Colour: 400 - 100% Polyester - Length: 50cm - Weight: 16mg.

DAY 1 from Panning Procedure (see protocol page) was followed. Amplification was not started this day.

OD600 measurements of titration culture were as follow:

Time after inoculation	OD 600nm
3h45	0.4984 => dilution 200-fold
6h20	0.1111
6h52	0.3191
7h02	0.4210
7h07	0.4567
7h12	0.4885

10mL LB+Tet were inoculated with a single ER2738 colony at 37°C with shaking. This culture will be diluted 100-fold the next day to amplify the unamplified panning eluates.

NEB Phage Display on Polyester and Silk - DAY 1 (Part 2)

Results titration unamplified phage eluates 1st round:

Dilution	Number of plaques
	Silk	Polyester
10	864	504
100	75	34
1000	16	3
10000	5	0

Therefore, the pfu of the unamplified phage eluates are:

• Silk: 75x(10^2)x(10^2) = 7.5 x 10^5 pfu/mL
• Polyester: 34x(10^2)x(10^2) = 3.4 x 10^5 pfu/mL



End of DAY 1 (steps 9-11) was performed.

Week 15th - 21st August

NEB Phage Display on Polyester and Silk - DAY 2

DAY 2 from Panning Procedure (see protocol page) was followed.

OD600 measurements of titration culture were as follow:

Time after inoculation	OD 600nm
2h51	0.3813
3h01	0.4980

NEB Phage Display on Polyester and Silk - DAY 3

Results titration amplified phage eluates 1st round:

Dilution	Number of plaques
	Silk	Polyester
10^8	>1000	>1000
10^9	148	130
10^10	10	21
10^11	2	5

Therefore, the pfu of the amplified phage eluates are:

• Silk: 148x(10^9)x(10^2) = 1.48 x 10^13 pfu/mL
• Polyester: 130x(10^9)x(10^2) = 1.3 x 10^13 pfu/mL



DAY 3 from Panning Procedure (see protocol page) was followed.

Dilution of the amplified eluate for the second panning was done using the following dilution factors and volumes:

• Silk: 74-fold dilution => ~3.38µL of amplified phage eluate added to ~246.5µL of TBST 0.5%
• Polyester: 65-fold dilution => 3.85%L of amplified phage eluate added to ~246.5µL of TBST 0.5%

OD600 measurements of titration culture were as follow:

Time after inoculation	OD 600nm
2h38	0.3811
2h57	0.6282 => dilution 20-fold
3h35	0.0688
4h16	0.2626
4h34	0.4569

OD600nm of overnight culture was 4.833. This culture was therefore diluted 200-fold to use as amplification cultures.

NEB Phage Display on Polyester and Silk - DAY 4

Results titration unamplified phage eluates 2nd round:

Dilution	Number of plaques
	Silk	Polyester
10	146	113
100	4	3
1000	2	1
10000	0	0

Therefore, the pfu of the unamplified phage eluates are:

• Silk: 146x10x5x(10^2) = 7.3 x 10^5 pfu/mL
• Polyester: 113x10x5x(10^2) = 5.65 x 10^5 pfu/mL



DAY 4 was performed.

OD600 measurements of titration culture were as follow:

Time after inoculation	OD 600nm
3h29	0.1474
3h53	0.3094
4h03	0.3862
4h09	0.4469
4h16	0.5200

NEB Phage Display on Wool and Linen - DAY 1

Wool used: Pure wool gauze twill fabric 100%wool 3.2 oz/linear yard- From Dharma trading.
Linen used: 100% bleached linen, 3.8 oz/linear yard- From Dharma trading.
A rectange of 0.5cm x 2cm was used (total surface: 2 cm² counting both sides).
Followed Day1 of the phage display protocol but some things to note -
During step7, I removed the fabric from the elution buffer and then I centrifuged the solution to remove the lint particles from the wool fabric and then neutralized the supernatant.
Note- Wool fabric used is loosely woven, so while washing it is coming apart, I did not have the same problem with linen.

Time after inoculation - Titration culture	OD 600nm
0	>0
2h 15	0.25
2h 35	0.43
2h 48	0.48

For amplification, 1:100 dilution of the overnight culture of ER2738 was used.

NEB Phage Display on Polyester and Silk - DAY 5

Results titration amplified phage eluates 2nd round:

Dilution	Number of plaques
	Silk	Polyester
10^8	>1000	>1000
10^9	152	150
10^10	14	15
10^11	1	3

Therefore, the pfu of the amplified phage eluates are:

• Silk: 152x(10^9)x(10^2) = 1.52 x 10^13 pfu/mL
• Polyester: 150x(10^9)x(10^2) = 1.50 x 10^13 pfu/mL



DAY 5 from Panning Procedure (see protocol page) was followed.

Dilution of the amplified eluate for the second panning was done using the following dilution factors and volumes:

• Silk: 76-fold dilution => ~3.3µL of amplified phage eluate added to ~246.7µL of TBST 0.5%
• Polyester: 75-fold dilution => 3.35µL of amplified phage eluate added to ~246.6µL of TBST 0.5%

OD600 measurements of titration culture were as follow:

Time after inoculation	OD 600nm
3h46	0.3272
4h01	0.4319
4h05	0.4869

NEB Phage Display on Wool and Linen - DAY 2

Results of 1st round unamplified phage eluate titration:

Dilution	Number of plaques
	Wool	Linen
10	>1000	>1000
100	87	357
1000	10	46
10000	0	1

Therefore, we have following phage concentration -
Wool - 87x(10^2)x(10^2) = 8.7 x 10^5 pfu/mL.
Linen - 357x(10^2)x(10^2) = 3.57 x 10^6 pfu/mL.
Followed Day2 protocol exactly.
OD measurements:

Time after inoculation	OD 600nm
0	0
3h30	2.42
Diluted 1:20
4h00	0.443
4h07	0.5409

Plaque amplification for Sequencing - Polyester and Silk

Plaque amplification protocol was followed. 20 plaques for each fabric were amplified.

NEB Phage Display on Wool and Linen - DAY 3

Results of 1st round amplified phage eluate titration:

Dilution	Number of plaques
	Wool	Linen
10^8	>1000	>1000
10^9	267	277
10^10	41	22
10^11	2	4

Therefore, we have following phage concentration -
Wool - 267x(10^9)x(10^2) = 2.67 x 10^13 pfu/mL.
Linen - 277x(10^9)x(10^2) = 2.77 x 10^13 pfu/mL.
Round 2 panning:
To get a final phage number per reaction approximately 2*10^11, add 10µl of the amplified phage solution in 240µl TBST(0.5% Tween 20) to get a final volume of 250µl for both wool and linen
Followed Day3 protocol of Phage display protocol.
OD measurement of Culture for Titration

Time after inoculation	OD 600nm
0	0
2h20	0.303
2h32	0.521
Diluted 1:5	Because of Hood availability
3h32	0.363
3h45	0.4759

OD measurements of Culture for Amplification

Time after inoculation	OD 600nm
	Wool	Linen
1h00	>0.0129	>0.009
1h25	0.0179	0.0139

NEB Phage Display on Wool and Linen - DAY 4

Results of 2nd round unamplified phage eluate titration:

Dilution	Number of plaques
	Wool	Linen
10	48	>1000
100	4	150
1000	1	18
10000	0	1

Therefore, we have following phage concentration -
Wool - 48x10x(10^2) = 4.8 x 10^4 pfu/mL.
Linen - 150x(10^2)x(10^2) = 1.5 x 10^6 pfu/mL.
Followed Day4 of the protocol exactly..
OD measurements:

Time after inoculation	OD 600nm
0	0
3h05	0.421
3h10	0.534

NEB Phage Display on Wool and Linen - DAY 5

Results of 2nd round amplified phage eluate titration:

Dilution	Number of plaques
	Wool	Linen
10^8	>1000	>1000
10^9	394	269
10^10	50	35
10^11	4	3

Therefore, we have following phage concentration -
Wool - 394x(10^9)x(10^2) = 3.94 x 10^13 pfu/mL.
Linen - 269x(10^9)x(10^2) = 2.69 x 10^13 pfu/mL.
Round 3 panning:
To get a final phage number per reaction approximately 2*10^11, add-
Wool - 5.1µl of the amplified phage solution in 244.9µl TBST(0.5% Tween 20) to get a final volume of 250µl
Linen - 10µl of the amplified phage solution in 240µl TBST(0.5% Tween 20) to get a final volume of 250µl
Followed Day5 protocol of Phage display protocol.
OD measurements:

Time after inoculation	OD 600nm
0	0
3h00	0.364
3h15	0.522

NEB Phage Display on Wool and Linen - DAY 6

Results of 3rd round unamplified phage eluate titration:

Dilution	Number of plaques
	Wool	Linen
10	499	>1000
100	38	547
1000	5	58
10000	0	6

Therefore, we have following phage concentration -
Wool - 38x100x(10^2) = 3.8 x 10^5 pfu/mL.
Linen - 58x(10^3)x(10^2) = 5.8 x 10^6 pfu/mL.


Week 22nd - 28th August

Purification of Phage DNA from Phage Display on Polyester and Silk for sequencing

Purification of phage DNA was carried out following the Purification of phage DNA protocol.
Note: The 500µL of phages R3P15 and R3P16 were by mistake mixed together. This was thrown away and subsequent purification was carried out with the remaining 250µL of phage stock.

Quantification was done using a Nanodrop2000. Phage DNA concentrations and purities were as follow:

	Polyester			Silk
Sample Number	Concentration (ng/µL)	260/280 ratio	260/230 ratio	Concentration (ng/µL)	260/280 ratio	260/230 ratio
R3P1	69	3.17	2.27	49	2.06	0.18
R3P2	50	2.77	0.69	66	2.95	2.25
R3P3	36.5	2.88	0.23	72	3.64	2.54
R3P4	47	2.28	0.21	79	2.34	1.24
R3P5	57	3.44	1.70	27	4.45	0.20
R3P6	84	3.66	3.91	65	4.04	2.39
R3P7	50	2.23	0.28	76	5.72	3.92
R3P8	59	2.06	0.20	25	3.02	0.10
R3P9	46	3.54	1.12	29	10.32	0.96
R3P10	54	2.54	0.60	30	9.51	0.98
R3P11	77	2.95	2.34	25	10.35	0.57
R3P12	74	3.07	2.29	56	3.68	1.81
R3P13	48	2.12	0.15	27	2.21	0.06
R3P14	29	3.09	0.14	38	9.76	1.55
R3P15	48.5	2.24	0.18	8	9.21	0.02
R3P16	64	2.13	0.45	44.5	33.87	2.14
R3P17	54.5	2.74	0.89	19.5	6.71	0.20
R3P18	50	2.19	0.21	27	6.04	0.52
R3P19	39	2.55	0.18	31.5	4.44	0.52
R3P20	59.5	3.53	1.78	42.5	3.74	1.16

Some of the samples (actually almost all of them) have weird 260/280 and 260/230 values. I do not know if this is going to be a problem for sequencing. To test, I will send only 5 samples (Samples R3P1 to R3P5 of the SILK experiment) to check that sequencing is OK.

Sequencing of phage DNA from NEB phage Display on Silk and Polyester

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


Sequences Phages NEB Silk
Sequences Phages NEB Polyester

Sequencing of Phage DNA from Phage Display on Wool and Linen

I proceeded with plaque isolation and Phage DNA isolation for sequencing. I picked 5 plaque each for R1 and R2 panning
unamplifies titration plates of wool and linen and 20 each for R3 panning unamplified titration plates of wool and linen.
Please refer following protocols from the protocol page:

• Plaque Amplification for ELISA or Sequencing - for plaque isolation and amplification and
• Rapid Purification of Sequencing Templates - for sample preparation for sequencing

DNA concentration and 260/280 ratio of purified templates for sequencing:

Linen_Round3 Panning	Concentration (ng/µL)	260/280 ration
R3P1	78	1.80
R3P2	12	3.12
R3P3	15	2.03
R3P4	22	2.45
R3P5	34	1.98
R3P6	18	2.27
R3P7	14	3.78
R3P8	21	1.78
R3P9	19	2.08
R3P10	19	2.76
R3P11	15	2.53
R3P12	19	1.92
R3P13	24	3.10
R3P14	15	2.48
R3P15	30	1.65
R3P16	79	8.36
R3P17	31	1.93
R3P18	23	1.93
R3P19	24	2.20
R3P20	25	1.75

Linen_Round1	Concentration (ng/µL)	260/280 ration
R1P1	35	2.73
R1P2	20	2.59
R1P3	28	2.40
R1P4	22	2.43
R1P5	26	2.51

Linen_Round2	Concentration (ng/µL)	260/280 ration
R2P1	27	2.06
R2P2	56	2.04
R2P3	66	2.06
R2P4	32	2.47
R2P5	25.8	2.05

Wool_Round3 Panning	Concentration (ng/µL)	260/280 ration
R3P1	94	1.88
R3P2	72	1.98
R3P3	68	1.94
R3P4	81	1.77
R3P5	82	1.85
R3P6	87	1.86
R3P7	80	1.85
R3P8	61	3.31
R3P9	73	1.85
R3P10	59	1.95
R3P11	68	1.85
R3P12	64	1.88
R3P13	57	1.93
R3P14	70	1.93
R3P15	69	1.87
R3P16	66	2.22
R3P17	63	1.88
R3P18	94	2.14
R3P19	73	2.03
R3P20	65	1.91

Wool_Round1	Concentration (ng/µL)	260/280 ration
R1P1	16	2.79
R1P2	26	4.00
R1P3	24	2.21
R1P4	19	2.15
R1P5	34	2.28

Wool_Round2	Concentration (ng/µL)	260/280 ration
R2P1	56	2.05
R2P2	45	2.00
R2P3	54	2.01
R2P4	52	2.08
R2P5	61	1.97

All Round 3 samples were sent to sequencing by GATC Biotech.
Results are available below:

NEB Phage Display on Wool and Linen - Finding a consensus sequence

Sequences were analysed using Geneious 9.0.2.
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 to sequence as the -28 seemed too close to the library sequence for the sequencing to give a good result.

Some samples (R3P5, R3P12, R3P17,R3P18 of wool and R3P3, R3P8,R3P11 of linen) did not exhibit those motifs. Deletion?
The 21 nucleotides of the library of remaining sequences were then translated.

Consensus sequence for Linen:



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 for Wool:



At 25% threshold-



NEB Phage Display on Polyester and Silk - Finding a consensus sequence

Sequences were analysed using Geneious 9.1.5.
As for the sequences obtained with cotton, 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 again used only the -96 primer (see sequencing guidelines scheme in protocol page) to sequence as the -28 seemed too close to the library sequence for the sequencing to give a good result.

Among the 20 plaques picked for polyester, one (R3P19) did not exhibit the randomized region (nor did the linker sequence and the end of the mature pIII protein coding sequence). Deletion?
This sequence was not used in the analysis.
Additionally, four of the sequences of phages selected on silk showed multiple randomized sequences:

• R3P9: 2 randomized sequences in the same orientation.
• R3P10: 3 randomized sequences with first and last one having the same orientation.
• R3P11: 2 randomized sequences in opposite orientation.
• R3P17: 2 randomized sequences in the same orientation.

Those sequences will be treated separately.
The 21 nucleotides of the library of the 19 remaining sequences for polyester and the 16 remaining sequences for silk were then translated.

Polyester:



Silk:



Using the Multiple-Align function of Geneious and setting the threshold for the consensus sequence at 0% (amino acid majoritarily present at a given position), we get the following consensus sequences:

Polyester:



Silk:



Week 29th August - 4th September

Spoke to one of our advisor - Ariel Lindner - He had some concerns about specificity and suggested few experiments.
This is what he suggested-

• For panning to find wool binding peptides- incubate every other fabric before wool, so that it removes any sticky peptides from the pool.
• He also suggested to do a panning with M13 phage without peptides to see the number of phage particles that bind.
• For later experiments at quantifying the binding affinity of the peptides for the target we should use enzymes that are easy to quantify using calorimeter like HRP and the likes instead of GFP, like this we hope to avoid the problem of autofluorescence from the target.

I started amplification of the 3rd round panning eluate of wool and linen- It would be Day 6 I guess..
OD measurements-

Time after incubation	OD 600nm
	Wool	Linen
0	0	0
1h	0.008	0.003
1h 45	0.0198	0.0186

Day 7 - Completed amplification and titred for both wool and linen.


Following the suggestion from Ariel-
Fourth round of panning - I started with Linen first. Plaques counted from the amplified R3 panning eluate-

Dilution	Number of plaques
	Wool	Linen
10^8	>1000	>1000
10^9	133	279
10^10	13	29
10^11	2	3

• Wool--> 133*10^9*10^2 =1.33*10^13 pfu/ml
  Linen-->279*10^9*10^2 =2.79*10^13 pfu/ml
  I tested two diferent parameters today-
  
  • The wash time, as a reaction's rate mainly depends on its rate of dissociation.
  • Panning with only M13 phage without peptides.. to check if these phages bind non specifically to the fabric.
• Calculation for Linen-
  2.79 *10^13 phages in 1000μl-
  To get final phage count as ~2*10^11--> 2*10^11/2.79*10^10=6.79μl.
  I decided to reduce the size of the fabric used to provide some sense of competition for the target - I used 1cm long fibre of the fabric- accordingly, I reduced the volume to 100μl so that all the phages can come in contact with the target.
  Also, to remove all the sticky peptide sequences, I incubated the phages first with 4 fabrics together- wool, silk, polyester, cotton.
  Note: There were more than usual number(0-3) of white plaques along with the blue ones in the titration plates.. Contamination?? I will be autoclaving all my solution just in case.
• The M13 phage concentration was 1.5*10^14 pfu/ml
  to get final phage count as ~2*10^11--> 2*10^11/1.5*10^11=2μl
• Fabric size-
  Silk- 10cm long thread
  Polyester- 10cm long thread
  Cotton - 0.25 cm2 square
  Wool- 0.25 cm2 square
  
• Day 8-Followed the day 1 of Phage display protocol with slight changes-
  • Washed the fabric/threads in ethanol/autoclaved water.
  • Blocked for 1h at 4 deg.
  • Incubated all fabric except linen in the phage solution for 1hr at room temperature.
  • Removed the fabrics, centrifuged and collected the supernatant.
  • Incubated linen (1cm fibre) with the remaining non sticky phages for 1h at room temperature.
  • Elute, neutralize and titre- this time I titred only 101 ,102,103 for different condition and two different phage type.

OD measurement for the titration culture:

Time after incubation	OD 600nm
0	0
3h	0.298
3h 30	0.61=> Diluted 2:1
3h 35	0.492

Week 5th September - 11th September

Week 12th September - 18th September

Week 19th September - 25th September

Week 26th September - 02nd October

Week 03rd October - 09th October

Week 10th October - 16th October

Week 17th October - 23rd October