, Infecter avec les stocks de phages obtenus en fin de production dilués dans du PBS : 10 µL de chaque dilution de phages ajoutés à 90 µL de bactéries TG1 à DO 600 0, vol.5

, Etaler les 100 µL sur des boites LB Ampicilline (100 µg/mL) 2% Glucose et laisser pousser O/N à 37°C

, Compter le nombre de colonies obtenues et le ramener pour obtenir un titre en nombre de colonies par mL

, ! Test de présentation par les phages

, ELISA : soit un ELISA fonctionnel adsorbé avec de la ?galactosidase, antigène sur lequel se fixe l'anticorps 13R4, soit un ELISA adsorbé avec un anti-Fab (référence I5260 Sigma-Aldrich, adsorbé à 1 µg/mL) pour un test de présentation de n'importe quel anticorps de la banque FabIg. La technique ELISA consiste à immobiliser un antigène ou une protéine sur un support (plaque 96 puits par exemple). Les phages qui présentent correctement le fragment d'anticorps seront révélés en fin d, La présentation des fragments d'anticorps par les phages peut être mesurée selon deux types d

, Dans notre cas, il s'agit de la ?galactosidase diluée en PBS à 10 µg/mL, 100 µL/puits en plaque 96 puits. Placer une feuille de parafilm entre la plaque et son couvercle pour éviter l'évaporation. L'incubation se fait sur la nuit à température ambiante

, Saturer la plaque au PBS lait 4% 200 µL par puits pendant 2 heures à température ambiante

!. Vider and L. Puits, Déposer les phages dilués en cascade pour avoir une dose-réponse

, ! Laver trois fois successives au PBS Tween 0,1% pour éliminer les phages qui ne reconnaissent pas l'antigène

, ! Incuber l'anticorps anti-M13 (qui cible la pVIII) couplé à la HRP et dilué au 1/5000 e en PBS lait 4% pendant 1h à température ambiante

!. Révéler, , p.100

, Stopper la réaction par ajout d'acide sulfurique (H 2 SO 4 1M, p.50

, ! Fragments Fab solubles : production et test de fonctionnalité Pour produire des fragments d'anticorps solubles, il faut travailler avec une souche bactérienne non suppressive qui reconnait partiellement le codon ambre comme un stop. Le fragment n'est ainsi pas fusionné à la protéine phagique pIII. En induisant une activité de synthèse accrue du promoteur, l'usage d'IPTG peut optimiser la production

, Il est nécessaire de travailler avec une souche non suppressive comme par exemple HB2151. Ces bactéries peuvent être infectées avec le stock de phagemides portant la séquence du fragment d'anticorps à produire. Après l'infection, les bactéries sont isolées sur boites afin d

. !-faire-une-préculture-o/n,

. Faire-une-préculture-de-jour,

, ! Ajouter l'IPTG à 1mM final

, ! Centrifuger à 4 000 rpm, 4°C pendant 15 minutes pour sédimenter les bactéries

, ! Récupérer le surnageant de culture contenant les protéines solubles sécrétées

, ! Congeler/décongeler à de multiples reprises le culot avant lyse

. !-resuspendre-le,

, NaCl 30mM ; Nonidet-P-40 0,65% (w/v) ; lysozyme de blanc d'oeuf de poule, vol.0, p.1

, mg/mL ; Benzonaze® Nuclease (Merck Milipore 4 U/mL)

, Incuber 1 heure en chambre froide sous agitation rotative délicate (pour éviter la mousse)

, ! Ajouter 100 mM de NaCl

, ! Centrifuger à 10 000 rpm, 4°C pendant 30 minutes

, Récupérer le surnageant qui correspond à la fraction soluble de protéines (périplasme)

. !-laver-le,

, ! Centrifuger à 13 000 rpm, 4°C pendant 10 minutes

!. Eliminer-le-surnageant,

. !-resuspendre-le-culot-en-pbs1x,

!. Stocker-À--20°c,

!. Facs,

, L'expérience de cytométrie en flux a été réalisée à partir de la lignée cellulaire HEK_FcIgG1 transfectée de manière transitoire ou stable avec les plasmides codant pour la GFP, la mCherry et le plasmide de Cre recombinase dans le cas de la transfection stable

, Après transfection et période de sélection en cas de transfection stable

, Ajouter la trypsine préchauffée à 37°C pour décoller et individualiser les cellules (1 mL pour un puits d'une plaque 6 puits) et incuber 15 minutes à 37°C

, Ajouter du milieu de culture pour diluer et stopper l'action de la trypsine

, ! Centrifuger 5 minutes à 1100 rpm pour sédimenter les cellules

. !-resuspendre-le-culot-en-pbs-pour and . Rincer,

, ! Centrifuger 5 minutes à 1100 rpm pour sédimenter les cellules

, Resuspendre le culot de cellules en PBS pour être à environ 500 000 -1 millions de cellules par mL (1 mL pour un puits de plaque 6 puits à environ 70-80% de confluence)

, ! Passer en tubes FACS, les couvrir d'une feuille d'aluminium pour les protéger de la lumière et conserver l'ensemble sur glace jusqu'à analyse

, L'analyse a été réalisée sur un cytomètre en flux Gallios (Beckman Coulter) et traitée avec le logiciel associé Kaluza

, ! Banque d'anticorps au format Fab (FabIg)

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, Use of M13KO7 Helper Phage for isolation of single-stranded phagemid DNA | NEB. ANNEXE Construction de la banque publiée dans, Methods in Molecular Biology, vol.1701, pp.239-253, 2018.

, Common Materials 1. 100 mg/mL ampicillin and/or carbenicillin in H2O (stock solution, see Note 1). Store at -20 °C

, mg/mL chloramphenicol in ethanol (stock solution). Store at -20 °C

, mg/mL kanamycin in H2O (stock solution). Sterilize by filtration and store at -20 °C

, 10 g/L of yeast extract, 5 g/L of NaCl, pH 7.0. In 900 mL of H2O, dissolve 16 g of Tryptone (Peptone), 10 g of yeast extract, and 5 g of NaCl

, 40% glucose solution. Sterilize by autoclaving. Store at RT

, LB agar plates: 10 g/L of Tryptone (Peptone), 5 g/L of yeast extract, 10 g/L of NaCl, 1.5 % agar

. Lb/ga-plates, Add 50 mL of 40% glucose solution and 1 mL of 100 mg/mL ampicillin in a final volume of 1L of LB agar medium, 2% glucose

. Lb/c-plates, Add 500 ?L of 30 mg/mL chloramphenicol in a final volume of 1L of LB agar medium, p.15

. Lb/gac-plates, Add 50 mL of 40% glucose solution, 1 mL of 100 mg/mL ampicillin, and 500 ?L of 30 mg/ml Chloramphenicol in a final volume of 1L of LB agar medium. 10. 10 14 pfu/mL KM13 helper phage (15) stock (see Note 2), 2% glucose, 100 ?g/mL ampicillin, 15 ?g/mL Chloramphenicol in LB agar medium

, PEG/NaCl solution: 20% (w/v) PEG, 2.5 M NaCl in H2O. Autoclave and store at 4 °C

, PBS x10: 1370 mM NaCl, 27 mM KCl, 43 mM Na2HPO4, 15 mM KH2PO4. In 900 mL of H2O

E. Z. M13-dna-mini and . Kit,

, NucleoSpin plasmid kit

, 50 mL conical centrifuge tubes (see Note 4) and a refrigerated centrifuge

, mL microcentrifuge tubes and a refrigerated bench-top centrifuge (see Note 5)

, Preparation of Uracil-Containing Single-Stranded DNA Template 1. E. Coli K12 CJ236: F?(HindIII)::cat (Tra + Pil + Cam R )/ ung-1 relA1 dut-1 thi-1 spoT1 mcrA (see Note 6)

, In the protocol below we will use pHEN1 phagemid vector (16) containing the scFv13R4, vol.17

, mg/mL Uridine stock in H2O, sterilize by filtration, and store at -20 °C

, 25 ?g/mL of Kanamycin, 100 ?g/mL of Ampicillin, 0.25 ?g/mL of Uridine (1.25 ?L of stock solution) in 2xYT medium

, Synthesis of the Mutagenized Complementary Strand 1. 100 ?M of the 5'-phosphorylated mutagenic primers in Tris-HCl pH 8 / 1 mM EDTA (Figure 1) (see Note 8)

, NEB2 x10 buffer:100 mM Tris-HCl (pH7.9 at 25 °C), 500 mM NaCl, 100 mM MgCl2, 10 mM DTT). Store at -20 °C

. 5u/?l-t4-dna-ligase,

, 10x ligase buffer: 400 mM Tris-HCl, 100 mM MgCl2, 100 mM DTT, 5 mM ATP

U. Polymerase,

E. Coli,

, 500 mL centrifuge bottles

, Sterile magnetic stir bars

, Magnetic stirrer

, 1M HEPES: weight 2.38 g of HEPES, add 8 mL of H2O

/. Glycerol and . Hepes:-1%-glycerol, Weight 10 g of glycerol, make up to 1 liter with water, autoclave. Add 1 mL of sterile 1M HEPES

, Add 1 mL of sterile 1M HEPES to 1 liter of autoclaved ultrapure water, H2O/HEPES: 1mM HEPES

, Electroporation and Phage Production 1. SOC medium: 20 g/L of tryptone (peptone), 5 g/L of yeast extract, 0.5 g/L of NaCl, vol.18

/. Kcl, 5 g of yeast extract, 0.5 g of NaCl, 10 mL of 250 mM KCl. Make up to 1 liter with water, 190.4 g/L MgCl2, 20mM glucose

, ml sterile polypropylene round-bottom culture tubes (17 mm x 100 mm)

G. , , vol.2

, Sterile Pasteur pipettes

, of Uracil-Containing Single-Stranded DNA Template In this protocol, we will first infect a dut ung strain with our phagemid; we then make a stock of phages using KM13 helper phage, and finally purify the single stranded DNA (ssDNA) encapsided in the phage particles. This ssDNA will contain uracil instead of thymine and, 2xTY/KC: 25 ?g/mL kanamycin and 100 ?g/mL carbenicillin in 2xYT medium

, Pick a single fresh colony of CJ236 in 2 mL of 2xYT and grow overnight (ON) at 37 °C with shaking

, Add 10 ?L of a pHEN1-13R4 phage stock diluted to 10 6 cfu/mL (see Note 11)

, Incubate 1 h at 37 °C without or with a slow shaking (0 -100 rpm)

, Plate 100 ?L on LB/GAC plates and incubate ON at 37 °C (see Note 12)

, Add 20 ?L of a 1/1000 th dilution of the KM13 helper phage (10 11 pfu/mL) (see Note 13)

, Incubate without shaking for 30 min at 37 °C (see Note 14)

, Resuspend the pellet in 30 mL of 2xYT/KAU (2xYT /kanamycin, ampicillin, uridine)

, Grow ON with vigorous agitation (220-240 rpm) at 37 °C in a 150 mL flask

, Remove the supernatant by inverting the tube with caution and put it gently upside down on absorbent paper to remove excess liquid (see Note 15)

, Spin briefly and remove the remaining liquid with a pipette and using absorbent paper as in the previous step

, Centrifuge at 4 °C for 5 min at 16,000 g in a bench-top centrifuge to pellet any insoluble material (see Note, vol.17

, Purify the ssDNA on 1 HiBind® M13 DNA Mini column (E.Z.N.A.® M13 DNA Mini Kit) by starting at step 4 (see Note, vol.18

, Pour a 1% agarose gel without any intercalating agent using TAE buffer (see Note 20)

, Incubate the gel for 30-60 min in a solution of TAE with 10 ?g/mL of SYBR Safe DNA Gel Stain (see Note 21). A typical result with some of the most frequent problems is shown in Figure 2

, Purify the DNA on a NucleoSpin Plasmid column using the "Plasmid DNA clean-up" procedure (see Note 26)

, Elute the DNA in 50 ?L of AE buffer (5 mM Tris/HCl, pH 8.5) heated to 70 °C to maximize yield (see Note 27)

. Optional, Run an agarose gel to check the efficiency of the second-strand synthesis

, Pour a 1% agarose gel without any intercalating agent using TAE buffer (see Note 28)

, Analyze 2 ?L of your mutagenesis in parallel with the same amount of ssDNA

, Incubate the gel for 30-60 min in a solution of TAE with 10 ?g/mL of SYBR Safe DNA Gel Stain (see Note 29). A typical result is shown in Figure 3

, Preparation of Electrocompetent Bacteria Use freshly prepared electrocompetent cells following the protocol below in order to obtain the high transformation efficiency (typically 5.10 9 -2.10 10 transformants/?g of supercoiled pUC18 plasmid) required for the final library transformation (see Note, vol.30

, All material must be pre-cooled and kept as close to 4 °C as possible in an ice/water bath throughout the preparation (see Note 31). If possible, work in a cold room. The centrifuge and the rotor must

, Pick a fresh colony of TG1 in a 50 mL flask containing 10 mL of 2xYT, and grow ON at 37 °C with vigorous shaking

, Pour the flask content in a 5 liter flask containing 1 liter of 2xYT, and grow at 37 °C with vigorous shaking (220-240 rpm) until OD600nm

, Pour the flask content in two 500 mL centrifuge bottles and cool down in an ice/water bath for 30 min. Mix regularly and gently the bottles

, Centrifuge at 5,000 g for 5 min at 4 °C and discard the supernatant

, Start with a vigorous stirring until the pellet detaches from the bottle; continue with a slower rotation rate until all the bacteria are completely resuspended. You may also gently mix the bottle by turning it upside down several times

, Centrifuge at 5,000 g for 10 min at 4 °C and discard the supernatant gently

. Resuspend, 50 mL of cold glycerol/HEPES. Pool the two bottles in a new centrifuge bottle. Do not transfer the stir bars, vol.6

, Centrifuge at 5,000 g for 15 min at 2 °C and discard the supernatant

, Resuspend the pellet in 1 mL of cold glycerol/HEPES using a cold 10 mL pipette. The final volume should be around 2 mL, vol.32

E. Coli, Electroporation and Phage Production If you prepared your own electrocompetent cells in section 3.3, you must immediately proceed and electroporate your DNA since transformation efficiency will decrease if cells are frozen. Each mutagenesis prepared in step 5 of section 5 will generate, in a single electroporation experiment, between 5 x 10 8 and 5 x 10 9 clones. In this protocol, we directly make the stock of phages that can be then used in phage display experiments. With the volumes used below

, Prepare one sterile 50 mL centrifuge tube for each DNA preparation (section 5) containing 12 mL of SOC and two 14 mL sterile polypropylene culture tubes containing 0.95 mL of SOC

, Cool on ice: 1 electroporation cuvette for each DNA preparation, and 1 for the positive control; the same number of sterile microcentrifuge tubes

, mix 350 ?L of competent cells and the purified ligation (35-40 ?L, prepared in step 5 of section 5). Do not pipet up and down to mix since this will warm the cells

, Be sure to put the sample at the bottom of the cuvette by gently taping the bottom of the cuvette on a flat surface, and avoid introducing bubbles. Quickly wet the cuvette and the cuvette slide with absorbent paper

, Apply an electric pulse using the following conditions: 2,500 V, 25 µF

, Immediately transfer the cells to one of the pre-warmed sterile 50 mL centrifuge tube containing 12 mL of SOC by washing the sample with 1 mL of outgrowth medium using a Pasteur pipette

, Add 1 ?L of a highly purified supercoiled pUC18 (10 pg/?L) plasmid to 40 ?L of competent cells in one of the pre-chilled microcentrifuge tube. Follow steps 5-8 but resuspend in 0.95 mL of SOC using the second 14 mL pre-warmed tube

, ?L of 10 -1 and 10 -2 dilutions of the positive control; 100 ?L of 10 -2 , 10 -3 , 10 -4 and 10 -5 dilutions of each 50 mL conical tube (containing 12 mL of SOC and transformed bacteria). flask containing 200 mL of 2xTY with 2% glucose and 100 ?g/mL Carbenicillin, vol.100

, Add 20 ?L of KM13 helper phage at 10 14 pfu/mL (20-fold excess)

, Incubate for 30 min at 37 °C without or with a slow shaking (0 -100 rpm)

, Incubate ON in a 2 liter flask at 37 °C with agitation (220 -240 rpm)

, Calculate the size of the library and the transformation efficiency using the series of dilutions plated in step 12 (see Note 35)

, Centrifuge for 30 min at 10,000 g at 4 °C and discard the supernatant

, Add 75 mL of PEG/NaCl for a second precipitation and proceed as before, pp.23-26

, resuspend the pellet in 80 mL of cold PBS with 15% glycerol (see Note 36)

, Centrifuge for 30 min at 10,000 g at 4 °C and recover the supernatant containing the phages

, Estimate phage concentration using UV absorbance with the formula: phages/mL = (A269nm -A320nm) x 10 13 (see Note, vol.37

, Aliquot in 50 ?L and store at -70 °C (see Note 38)

, Carbenicillin and Ampicillin can be alternatively used. However, since it is more stable, we prefer to use Carbenicillin for the last step of the library production (section 3.4)

, We use here KM13 helper phage that confers resistance to kanamycin. M13KO7 or another helper phage can be alternatively used

, PEG 8000 for molecular biology from Sigma #81268)

, Be sure that the tubes are resistant enough. Falcon (#352070) and Corning (#430290) branded 50 mL polypropylene conical centrifuge tubes are resistant to 16, vol.000

, Phages are very stable even at high temperature but the expressed scFv are heat-sensitive

E. Coli, K12 CJ236 can be obtained from NEB. Streak out the strain on LB agar containing chloramphenicol (15 ?g/mL) to ensure that you start with an F + host, but do not include chloramphenicol in liquid media

, The protocol is flexible enough to work with any antibody format (scFv, Fv, Fab, (Fab')2, VHH, etc.), but requires a phage or phagemid vector. The mutagenic oligonucleotides

, High quality oligonucleotides must be used. The best is to order cloning-quality 5'-phosphorylated oligonucleotides

, TG1: supE thi-1 ?(lac-proAB) ?(mcrB-hsdSM)5 (rk -mk -)

, For phage display it is critical to check for F' presence. For this reason you must keep TG1 on a synthetic plate without proline (proAB), for instance M9 plates with glucose and thiamine (thi-1). Use a recently streaked plate of less than 1 week

, We use a large excess of bacteria to ensure that all the phages can infect a bacterium

, We use glucose in all the plates because the scFv is under the control of the lac promoter in pHEN1. This ensures a strong repression of the gene and avoid toxicity, pp.100-1000

, should have around 5 x 10 8 bacteria/mL. A 2-fold excess of phages is used, that is 10 9 KM13 per mL of culture. This is much lower than the classical excess of 10-20-fold used in most protocols

, Empty the supernatant into a liquid trash by inverting the tube, then

, Clean the hood with phagospray and use filtered pipette tips to prevent contaminations with filamentous phages

, The pellet may be absent since it is essentially due to bacteria that were not fully eliminated by the first centrifugation step, p.16

, To obtain the 50-70 ?g of ssDNA necessary for one or two mutagenesis, it is necessary to apply on the column around 10 13 phages in a maximal volume of 1.4 mL. Depending on the vector, this amount of phages is obtained from 30 -400 mL of culture. Alternatively, phenol extraction and ethanol precipitation can be used

, When using a 1 cm path length, a 33 ?g/mL solution of single-stranded DNA has an absorbance of 1 at 260 nm. The yield should be around 75 ?g

, Intercalating agents change the DNA supercoiling state and the migration speed. Resolution of single and double-stranded DNA is much better in their absence. For 1 liter of TAE x50: Tris 242 g, 57.1 mL acetic acid

, SYBR Safe DNA Gel Stain (Thermofisher), Ethidium Bromide

, We usually use a single mutagenic oligonucleotide for the CDR1s, CDR2s and the VL-CDR3, and a series of oligonucleotides of different lengths for the VH CDR3. We prepare one mix for each VH-CDR3 length (10 ?L of each oligonucleotide) to ensure an equal representation of the CDR3 lengths in the library

, pHEN1-13R4 vector is 5229 bases long. If your phagemid contains N bases, 15 pmoles of ssDNA represents

, It is extremely important to cool down very quickly from 90 °C to 4 °C to avoid a hybridization bias due to a partial matching between the degenerated oligonucleotide and the original CDR sequence. You can use a thermal cycler or simply boil your sample and transfer, p.17

, This is a very high dNTP concentration, much higher that what is used in most protocols (0.1 -0.6 mM)

, You can also heat-inactivate the reaction and purify by precipitation

, See section 2.5 "Elution procedures" in the Macherey-Nagel manual

, Intercalating agents change the DNA supercoiling state and the migration speed. Resolution of single and double-stranded DNA is much better in their absence. For 1 liter of TAE x50: Tris 242 g, 57.1 mL acetic acid

, SYBR Safe DNA Gel Stain (Thermofisher), Ethidium Bromide

, You can contact them to get bulk quantities (10 x 400 ?L). cutting the tip at around 5 mm from the extremity

, the Biorad technical note MC1652101C

, If the library is not large enough and the transformation efficiency lower than 5 x 10 9 you must improve electrocompetent cell preparation or use commercial ones

, Check the efficiency of this step by analyzing your sample on an agarose gel: no ssDNA but a strong dsDNA band should be visible

, You can scale it down or up depending on the anticipated results (see, Notes, vol.37

, It is better to also titer the infectious phages using serial dilutions, infection of mid-log TG1, then plating on LB/GA (cfu/mL). In general, The formula depends on the phage/phagemid size

, Each aliquot should be a hundred times larger than the library size measured by titration. For most libraries this means around 10 12 phages

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