Protocol for S100 phage display experiments
Luke Wheeler
(Also see Luke Wheeler Notebook #9 for lab notes)
Necessary materials
NEB PhD-12 kit phage library
Purified S100 protein
EZ-link BMCC biotin
Pierce high binding capacity streptavidin plate (or similar)
Loading buffer (TBST or similar)
PBS (for biotin-labeling reaction)
Sterile PCR tubes
Sterile 1.5mL tubes
Sterile ddH2O
10mM biotin stock from NEB PhD-12 kit
PhD kit blocking buffer (see NEB manual)
PEG/NaCl (see NEB phage booklet)
Top agar (see NEB phage booklet)
XGAL/IPTG agar plates
Filter tips (20uL, 200uL, 300uL)
ER2738 E. coli cells
10% bleach bath (in beaker)
Sterile polystyrene plate
LB media
Cs1 Forward primer (5’-ACACTGACGACATGGTTCTACAGTGGTACCTTTCTATTCTCACTCT-3’)
96seq-Cs2 reverse primer (5’-TACGGTAGCAGAGACTTGGTCTCCCTCATAGTTAGCGTAACG-3’)
Barcoded P5/P7 secondary amplification primers
Preparation of loading buffer
Prepare the loading/wash buffer according to your desired conditions. I use a “TeBST” buffer: 50mM TES, 150mM NaCl, 0.1% Tween-20 as the base for all my buffers. For Ca2+ buffers (Ca-TeBST) add CaCl2 to the base buffer to a final concentration of 2mM. For EDTA elution buffer (EDTA-TeBST) add EDTA to a final concentration of 5mM. For loading buffers with competitor peptide, I dissolve the peptide directly into the Ca-EDTA buffer.
Protein biotinylation
Make sure there is only on Cys residue in protein (preferably at one of the termini). Label purified S100 protein with EZ-link BMCC biotin according to manufacturer protocol/specifications. Reduced the sample before labeling with TCEP while degassing (via VP-ITC vacuum pump). Then exchange into PBS and perform the labeling reaction in this buffer. Exchange into desired buffer after labeling is complete. Confirm labeling using the MALDI-TOF (should be a ~540 Da shift relative to unlabeled protein). I make a final stock of 10uM labeled protein in loading buffer, so that I can add 1uL in the preparation of phage samples
Phage display panning experiment
*PERFORM ALL STEPS INVOLVING PHAGE SAMPLES WITH FILTER TIPS*
1) Block desired number of wells on high-capacity streptavidin plate with PhD kit blocking buffer. Incubate for at least one hour in refrigerator.
2) While blocking plate, prepare samples of protein and phage in the desired loading buffer in sterile 1.5 mL tubes. Mix 98uL loading buffer, 1uL of 10uM biotinylated protein (or biotin for negative control) (final concentration = 0.1uM), and 1uL of NEB PhD-12 kit phage library (2.5 x 1010 PFU total). Mix gently and spin with microfuge gently. Incubate these solutions at room temperature for 2 hr.
3) As samples near end of incubation, wash the wells of the streptavidin plate with the desired loading/wash buffer. 5 washes with 150uL/wash. Pipette buffer onto well, let sit momentarily, then remove by pipetting. Be careful never to cross-contaminate any wells with different buffers. Leave plate wells covered in buffer until just before applying samples.
4) After samples are done incubating at room temperature, carefully transfer each 100uL sample to a single well of the streptavidin plate using a separate filter tip for each sample. Place in center of room temperature shaker and secure with tap. Rotate at 25 rpm for 20 min. to allow biotinylated proteins to bind to streptavidin. Then add 1uL of 10mM kit biotin to each well and incubate with 25 rpm shaking for an additional 5 min.
5) After incubation on plate is complete, carefully remove each solution from its respective well. Discard directly into 10% bleach bath, unless you have a reason to keep these samples (i.e. to titer for PFU/mL). Move on to the washing steps immediately after removal of samples. Never give the wells time to dry out.
6) Use a multichannel pipette with 300uL filter tips to wash the sample wells that contain bound protein/phage complexes. (I will have distributed >200uL aliquots of the loading/wash buffers into the wells of a sterile polystyrene plate for ease). Use the multichannel pipette to gently apply 200uL of appropriate wash buffer (i.e. Ca-TeBST, or containing a peptide competitor) to each well, incubate for ~5 sec. (as little time as possible), then carefully remove the buffer by slightly tilting the plate and pulling it away using the same set of filter tips. Discard these directly into the 10% bleach bath. Repeat this procedure for a total of 5 washes. Move on to elution step immediately after completing washes.
7) Use sterile filter tips to gently apply 100uL of EDTA-TeBST elution buffer to each well (can do this with the multichannel too if desired). Make sure never to cross-contaminate wells with phage from another well. Place the lid back on the plate, transfer to room temperature shaker, incubate for one hour while shaking at 25 rpm. This will allow time for the proteins to release bound phage into solution.
8) After the 1 hr. elution is complete, remove streptavidin plate from shaker. Gently remove the 100uL eluates from plate using sterile filter tips and transfer to fresh sterile 1.5 mL tubes. Store these eluates at 4°C. They will hold the same titer for about two weeks. However, I consider it best to move onto the next step ASAP.
Phage titering
Follow the instructions in the NEB PhD-12 kit instructions carefully to titer phage. Briefly: while doing the panning experiment grow a culture of ER2738 bacteria to mid log phase. Prepare serial dilutions of the eluates (I start by diluting 1uL from the 100uL sample into 9uL of LB and use a multichannel pipettes to do serial dilutions in LB in sterile PCR strip tubes). Use 10uL of desired dilutions to inoculate 200uL aliquots of mid log-phase ER2738 in sterile 1.5 mL tubes. Let incubate for a few minutes at room temperature. Then add each 210uL sample to 3mL of pre-melted top agar (see PhD-12 kit manual for recipe). Apply the entire mixture to a warm XGAL/IPTG agar plate. Let cool for approximately 5 min. at room temperature to solidify. Then invert and transfer to warm room overnight. The next morning, plaques should be visible (blue on XGAL/IPTG plates, although I have noticed a propensity for some phage to lose the ability to make blue plaques while still retaining randomer sequences). Count the plaques and calculate PFU/mL to quantify phage elution yield.
Phage amplification
If you will be continuing with further rounds of panning, follow the NEB PhD-12 kit manual instructions carefully to amplify the eluted phage in ER2738 culture and isolate them. Titer the amplified eluates to quantify PFU/mL before repeated the panning procedure as describe above.
Preparation of Illumina sequencing library
Preparing the Illumina library requires a two-step PCR. First the ssDNA of phage eluates is isolated. This ssDNA is then used as the template for a PCR reaction with primers that anneal to the phage genome outside of the randomer region. This product is purified and used as the template for a second PCR that adds barcodes and Illumina adapters to the product. These final products can then be quantified and mixed together for a multiplexed sequencing library.
1) Use the Qiagen M13 spin kit to purify the ssDNA from isolate phage pools. This kit has been officially discontinued, but is identical to the Qiagen mini-prep kit except for the phage precipitation buffer (bufffer MP), which can be made by dissolving 3.3 g citric acid monohydrate in 3 mL ddH2O. The manual for this kit can still be found online. Columns are the same as for mini-prep and can be ordered separately.
2) I measure the concentration of the purified phage ssDNA using the ssDNA_1mM protocol on our Eppendorf Biospectrometer. You will need 100ng of this DNA ( a ~7200 bp phage genome) for the primary PCR. I repeat these reactions 2-3 times and pool the products to help alleviate potential issues due to statistical sampling from the phage eluate ssDNA stock. Primer sequences are shown above. The forward primer anneals adjacent (just upstream) of the randomer region. The reverse primer anneals ~100 bp downstream at the binding site for the Phd-12 kit 96-seq Sanger sequencing primer (see manual).
3) Peform PCRs as follows: (for 25uL total volume)
Q5 buffer 5uL
10mM dNTPs 1uL
10uM Cs1 forward 1.25 uL
10uM 96seq-Cs2 reverse 1.25 uL
Template V to reach 100ng
Sterile ddH20 V to reach 25uL
Q5 polymerase 0.25uL
The thermal cycler settings for this PCR are as follows (and encoded as “phageamp1” in our gradient cycler):
a) 95°C hold (hot start)
b) 95°C 15s
65°C 15s
72°C 30s
c) 95°C 10s
72°C 15s
72°C 30s
d) 72°C 5 min.
e) 4°C hold
Repeat (b) for 5 cycles and (c) for 30 cycles
4) Run these PCRs (entire reactions) on a 2% agarose gel until good separation is achieved. There should be one main product around 220 bp (compare to a ladder to be certain). Excise the appropriate bands and purify DNA using a gel extraction kit. Quantify using dsDNA 1mm measurement.
5) Next, you will use the primary PCR products as a template for the secondary PCR, which adds barcodes and Illumina adapters. Choose a unique forward/reverse barcode pair for each sample. We have 8 forward primers (A1-A8) and 12 reverse primers (H1-H8), giving a total of 96 possible combinations. The sequences can be found in the excel spreadsheet “CS_indexing_oligos.xlsx”. You will need 50ng primary product as template for secondary PCR. Peform PCRs as follows: (for 50uL total volume)
Q5 buffer 10uL
Q5 GC enhancer 8uL
10mM dNTPs 2uL
10uM Cs1 forward 4uL
10uM 96seq-Cs2 reverse 4uL
Template V to reach 50ng
Sterile ddH20 V to reach 50uL
Q5 polymerase 0.5uL
The thermal cycler settings for this PCR are as follows (and encoded as “phageamp2” in our gradient cycler):
a) 95°C hold (hot start)
b) 95°C 5 min. (to ensure denaturation)
c) 95°C 15s
65°C 15s
72°C 15s
d) 72°C 30s
e) 10°C hold
Repeat (c) for 15 cycles
6) Run these PCRs (entire reactions) on a 2% agarose gel until good separation is achieved. There should be one main product around 289 bp (compare to a ladder to be certain). Excise the appropriate bands and purify DNA using a gel extraction kit. Quantify using dsDNA 1mm measurement. These should be the final products. I like to submit several (up to 11 on a single run) for fragment analysis to ensure that the products are the correct size. Distribution should have a sharp peak around 290 bp. Often there is a broader shoulder skewed to the right (larger), of which I do not know the origin. However, it has not proved to be a problem in our sequencing runs. We use rigorous quality control on the raw data. I mix the samples together according to mass, this has worked well to get the desired distribution of reads (i.e. if I want all samples to have the same number of reads I make a mixture with an equal amount of mass from each sample). When submitting to sequencing core, I err on the side of caution and submit a more concentrated library than required (10nM is required, so I overshoot and submit 50nM or higher. The facility can adjust after they correct the library concentration using qPCR).
Files
[embeddoc url=”https://harmslab.uoregon.edu/files/2013/05/CS_indexing_oligos-tgy6ic.xlsx” download=”all” viewer=”microsoft” ]