Phage display technology studies RNA binding protein experiments
Phage display technology is to fuse an exogenous gene with the surface protein gene of the phage, and the corresponding exogenous protein will be expressed on the surface of the phage particle fused with the exogenous gene, i.e., the protein encoded by the exogenous gene is displayed on the surface of the phage particle. This experiment is from "RNA Laboratory Guidebook", edited by Xiaofei Zheng.
Operation method
Phage display technology to study RNA-binding protein experiments
Principle
Phage display technology involves the fusion of an exogenous gene with the surface protein gene of a phage, and the corresponding exogenous protein is expressed on the surface of the phage particle fused with the exogenous gene, i.e., the protein encoded by the exogenous gene is displayed on the surface of the phage particle.
Materials and Instruments
Carrier tRNA Antibody Oligonucleotide Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
Antibiotic Storage Solution BCIP Glucose Storage Solution X-Gal Magnetic Bead Wash Protease Inhibitor RNase Inhibitor TENT Binding Buffer Blotting Buffer Bovine Serum Albumin Khomas Brilliant Blue Stain Acrylamide Gel Preparation Buffer Electrophoresis Buffer Sodium Acetate
Top Agar LB YT Paramagnetic Beads and Racks Dry Gel Device and Cellophane Nucleic Acid Electrophoresis Device In Vitro Transcription Kit
Distilled deionized water was used in all operations.
1. Vector: pDISPLAYblue B; host bacterium: DH12S (Gibco BRL Company); helper phage: M13K07 (Gibco-BRL Company).
2. Antibiotic storage solution (1000X): ampicillin (100 mg/ml, aqueous solution); kanamycin (70 mg/ml, aqueous solution); methicillin (200 mg/ml, aqueous solution). Filtered and sterilized, dispensed and stored at -20℃.
3. Culture solution/base:
LB: 10 g peptone, 5 g yeast extract, 10 g NaCl, add water to 1 L, add 15 g agar powder when spreading, autoclaved.
Top layer agar: 7 g agar powder per liter of LB culture solution, autoclaved.
2X YT: 16 g peptone, 10 g yeast extract, 5 g NaCl, add water to 1 L, autoclaved.
4. Add reagents to culture solution:
BCIP: 5-bromo-4-chloro-3-indolyl phosphate (5-bromo-4-chloro-3-indolyl phosphate, Sigma), dissolved in dimethylformamide, formulated into 40 mg/ml storage solution, stored at -20°C.
Glucose storage solution: 20% (m/V) aqueous glucose solution, filtered for sterilization.
X-Gal: 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (5-bromo-4-chloro-3-indolyl-β-D-galactopy-ranoside), dissolved in dimethylformamide, formulated as a 40 mg/ml stock solution, stored at -20°C.
5. Bead wash solution 1: 0.1 mol/L NaOH, 0.1 mol/L NaCl.
Bead Wash 2: 0.1 mol/L NaCl.
6. Paramagnetic Beads and Magnetic Racks: Dynabeads M-280 Streptavidin Magnetic Beads (Dynal).
7. PEG/ammonium acetate: 20% PEG8000, 3.5 mol/L ammonium acetate.
8. protease inhibitor:
Phenylmethylsulfonyl fluoride (PMSF): 100 mmol/L (dissolved in ethanol) storage solution, stored at -20°C, working concentration is 500 μmol/L.
Pepstatin: 1 mg/ml (dissolved in methanol) storage solution, stored at 4°C, working concentration is 0.7 μg/ml.
Lcupeptin: 1 mg/ml (dissolved in water) storage solution, stored at -20℃, working concentration is 0.5 μg/ml.
9. RNase inhibitor (RNasin, Promega).
10. TE: 10 mmol/L Tris-HCI (pH 8.0), 1 mmol/L EDTA, autoclaved.
11. TENT binding buffer: 10 mmol/L Tris-HCl (pH 8.0), 1 mmol/L EDTA, 250 mmol/L NaCl, 0.5% (V/V) Triton X-100.
12. tRNA: E.coli tRNA, 20 mg/ml aqueous solution.
13. antibody:
Primary antibody: anti-MYC labeled Hangman; anti-histidine antibody (Qiagen).
Secondary antibody: alkaline phosphatase labeled sheep anti-mouse IgG.
14. 10X blotting buffer: 250 mmol/L Tris base, 1.9 mol/L glycine, 1% SDS. 100 ml of 10X blotting buffer with 700 ml of water and 200 ml of methanol to make 1 L of blotting buffer.
15. Bovine Serum Albumin: Component V (Amersham).
16. Kaumas Brilliant Blue Stain: 0.2% (m/V) Kaumas Brilliant Blue R (Sigma) dissolved in 45% methanol, 9% acetic acid in water.
17. dry glue device and cellophane: protein electrophoresis and electrotransfer device; protein electrophoresis molecular mass marker (Bio-Rad); nitrocellulose membrane.
18. acrylamide gel preparation buffer:
2 mol/L Tris-HCl ( pH 8.8 ), 1 mol/L Tris-HCl (pH 6.8), 10% SDS.
30% Methylenedioxyacrylamide-acrylamide solution: 0.8:29.2 ratio of methylenedioxyacrylamide to acrylamide.
10% aqueous ammonium persulfate solution ( freshly prepared), tetramethylethylenediamine (TEMED), water-saturated n-butanol.
19. 2X Sampling Buffer Mix: 125 mmol/L Tris-HCl (pH 6.8), 10% SDS; 20% glycerol; 0.02% Bromophenol Blue. Add 900 μl of 2X Sampling Buffer Mix and 100 μl of β-mercaptoethanol per 1 ml of 2X Sampling Buffer before use.
20. Azabenzotetrazole/5-bromo-4-chloro-3-indole-phosphate (NBT/BCIP) mixture: 66 μl NBT solution [50 mg/ml NBT dissolved in 70% ( V/V) dimethylformamide] and 41 μl BCIP solution (40 mg/ml NBT in dimethylformamide) were added to 10 ml AP buffer [100 mmol/L Tris-HCl ( pH 9.0)]. Tris-HCl (pH 9.5), 100 mmol/L NaCl, 5 mmol/L MgCl2 ].
21. 10X electrophoresis buffer: 250 mmol/L Tris base, 1.2 mol/L glycine, 1% SDS.
22. TBST: 10 mmol/L Tris-HCl ( pH 8.0 ), 150 mmol/L NaCl, 0.05% ( V/V ) Tween 20.
23. biotin-labeled oligonucleotides: oligonucleotides biotinylated at the 5' end complementary to the 3' end of the RNA, pre-purified by gelatinization to remove unbiotinylated oligonucleotides to ensure that all annealed RNA binds to streptavidin beads.
24. Chloroform:isoamyl alcohol mixture: 49:1 chloroform:isoamyl alcohol.
25. nucleic acid electrophoresis apparatus; molecular mass marker for nucleic acid electrophoresis.
26. ethanol, formamide (divided into small tubes and stored at -20°C).
27. Electrophoresis gel preparation solution:
10X TBE: 0.89 mol/L Tris base, 0.89 mol/L boric acid, 0.02 mol/L EDTA.
30% (m/V) acrylamide/methylenedioxymethacrylamide solution, 19:1 ratio of acrylamide to methylenedioxymethacrylamide.
10% aqueous ammonium persulfate solution (freshly prepared).
Tetramethylethylenediamine (TEMED).
Urea.
28. In vitro transcription kits: T7 polymerase transcription kit (MEGAshortscript kit), SP6 polymerase transcription kit (MEGAscrip kit), both purchased from Ambion.
29. Nucleic acid electrophoresis molecular mass marker: ψX174DNA/HaeIII cut fragment.
30. 3 mol/L sodium acetate, pH 5.2.
31. Sephadex G-50: hydrated with TE (Sephadex G-50 dry powder was purchased from Pharmacia Biotech).
II. Methods of operation
When using phage display technology to study RNA-BP, we should first examine whether RNA-BP can be displayed by phage, and then test the function of the protein displayed on the surface of phage, based on which, we will use the phage display RNA-BP mutant library to screen and get the positive clones of RNA-BP that can bind to the target RNA.
1. Construction and screening of display recombinants
First of all, the gene fragments to be inserted into the vector should be determined. The entire RNA-BP gene can be inserted into the vector, or only the fragment containing the RNA-binding domain can be selected. Care should be taken to maintain the correct reading frame, not to contain stop codons, and to avoid containing charged amino acids close to the cleavage site of the vector signal peptide sequence. The RNA-BP encoding gene can be cloned by high-fidelity PCR, and the enzymatic sequence corresponding to the vector polyclonal site can be introduced into the primer sequence. Recombinants containing the exogenous gene are constructed by digestion and ligation.
Despite the presence of the lacⅠa gene in the host bacterium, there may still be a low level of transcription from the lac promoter. Adding 2% glucose to the culture medium can help to inhibit transcription of the lac promoter, so glucose should be added to the culture medium except for protein demonstration.
After the recombinant is constructed, the expression of the alkaline phosphatase-exogenous protein fusion protein can be examined on LB plates containing 100 μg/ml ampicillin, 40 μg/ml BCIP and 2% glucose. The host bacteria were transfected with pDISPLAY-blue-B and pDISPLAY-B (a vector in which the phoA gene encoding E.coli alkaline phosphatase was removed from the pDISPLAYblue-B vector by NotⅠ cleavage followed by ligation) as controls. pDISPLAYblue-B transfected host bacteria were blue clones, and pDISPLAYblue-B transfected host bacteria were blue clones, and pDISPLAYblue-B transfected host bacteria were blue clones. After overnight incubation at 37℃, pDISPLAYblue-B transfected host bacteria should be blue clones, pDISPLAY B transfected host bacteria should be white or very light blue clones (endogenous phoA gene expression is very weak and will not interfere with the color of the clones), and the color of the clones with the insertion of the exogenous gene should be in between. For more precise quantification of phoA expression, cell extracts can be used. In this assay, only the expression of the gene is needed to determine the transport of RNA-BP, i.e., as long as the color of the clone is slightly darker than that of the negative control, it indicates that the RNA-BP can be transported. If the transport is not effective, it can be adjusted by lowering the temperature. The plates can be incubated overnight at a lower temperature and then incubated at 37°C to detect phoA gene expression.
After determining the expression conditions of the recombinant, the phoA gene can be removed from the vector by NotⅠ cleavage and then ligated, so that the exogenous gene can be fused with gene Ⅲ to form pDISPLAY-B recombinant, which can be used for phage display in the next step.
2. Preparation of auxiliary phage
Bacteria containing the pDISPLAY-B recombinant must be infected by an auxiliary phage for phage display.
(1) Inoculate fresh E. coli host bacteria into 2 ml LB culture medium and incubate at 37°C with shaking (250 r/min ) overnight.
(2) Dilute the overnight culture 1:50 into 10 ml 2X YT culture medium and shake at 37°C for 30 min at 250 r/min. Meanwhile, dilute the overnight culture 1:500 into LB culture medium and shake at 37°C for 6 h at 250 r/min for detection of auxotrophic phage titer.
(3) Add auxin phage MI3K07 into 2X YT culture with a final concentration of 106 pfu/ml, and shake at 37°C 250 r/min for 30 min.
(4) Add kanamycin to a final concentration of 70 μg/ml, and continue shaking for 4 h at 250 r/min at 37°C.
(5) Add 1.3 ml of culture medium into each 1.5 ml centrifuge tube, centrifuge at 15000 g for 5 min, take 1 ml of supernatant from each tube into a clean centrifuge tube and centrifuge again.
(6) Take 0.9 ml of supernatant from each tube to a clean centrifuge tube and heat at 65℃ for 10 min to kill the residual bacteria, and the resulting auxin phage storage solution can be stored at 4℃ for several months.
(7) Dilute the phage reservoir solution with LB 4 times 1:100 (5 μl→495 μl), take 100 μl of 10-6 and 10-8 dilution, add 200 μl of each to the host bacterium used for detection of auxiliary phage titer, then add 3 ml of top layer agar (at 50°C), mix quickly, and then spread on the preheated LB plate at 37°C. At the same time, use the host bacterium and the auxiliary phage reservoir solution individually, and add 3 ml of the top layer agar (at 50°C) to the host bacterium, and then add 3 ml of the top layer agar (at 50°C), mix rapidly. At the same time, the host bacteria and phage were used separately as control.
(8) After the top layer of agar solidified, incubate the plates at 37°C until phage spots appeared.
(9) Count the number of phage spots on the plate and calculate the titer. Titer (pfu) = number of phage spots on plate X dilution X 10.
(10) Plates should be free of phage plaques for host bacteria alone and free of bacterial clones for phage alone.
(11) The titer of auxotrophic phage should preferably be between 1011~2X1011 pfu. If necessary, the phage reservoir can be precipitated with PEG to increase the titer.
3. Preparation of Display Phages
(1) Inoculate the host bacterium transfected with pDISPLAY-B recombinant into 2 ml of LB (containing 100 μg/ml ampicillin and 2% glucose) culture medium, and incubate overnight at 37℃ with 250 r/min shaking.
(2) Dilute the overnight culture 1:200 to 20 ml 2X YT culture medium (containing 100 μg/ml ampicillin and 2% glucose), and incubate at 37℃ with 300 r/min shaking until the OD600 reaches 0.5 (about 3~5 h).
(3) Take 15 ml of cultured bacteria and add it into a 50 ml tube, add auxiliary phage M13K07, the final concentration is 6 X 106 pfu/ml, and shake at 37℃ 250 r/min for 30 min.
(4) Centrifuge at 15000 g for 10 min at room temperature.
(5) Aspirate the supernatant, resuspend the bacterial precipitate with 30 ml of 2X YT culture medium (containing 100 μg/ml ampicillin and 70 μg/ml kanamycin), and incubate overnight at 30°C with 300 r/min shaking.
(6) Simultaneously inoculate the host bacteria into LB culture medium and incubate overnight for titer determination.
(7) Dilute the overnight cultured host bacteria for titer determination 1:500, and shake at 250 r/min for 6 h at 37°C [Step 13 below].
(8) Centrifuge at 3000 g for 20 min at 4°C to collect the phage culture solution from the overnight culture, take 16 ml of the supernatant, add 4 ml of PEG/ammonium acetate solution, turn and mix well, and then take an ice bath for 1 h. The phage culture solution will be used for the titer assay.
(9) Centrifuge at 15000 g for 15 min at 4°C and remove the supernatant.
(10) Resuspend the sample with 1 ml of TE containing protease inhibitor, transfer to a 1.5 ml centrifuge tube, centrifuge at 15000 g for 2 min, transfer the supernatant to a clean centrifuge tube, add 250 μl of PEG/ammonium acetate solution, mix well and then ice bath for 1 hour.
(11) Centrifuge at 15000 g for 15 min at 4℃ and remove the supernatant.
(12) Resuspend the sample with 100 μl of TE containing protease inhibitor. Store this display phage reservoir at 4°C (for several months) until titer measurement and immunoassay are completed before proceeding further.
(13) Dilute the display phage reservoir solution with LB 6 times 1:100 (5 μl→495 μl), take 100 μl of 10-8, 10-10 and 10-12 dilutions respectively, add 100 μl of each to the host bacterium used for the titer assay [obtained from the step (6)], and at the same time, use 100 μl of the host bacterium and 100 μl of the phage (1:100 dilution) as the control separately. At the same time, 100 μl of host bacteria and 100 μl of phage (1:100 dilution) were used as control, and LB plate (containing 100 μg/ml ampicillin and 2% glucose) was coated after incubation at 37℃ for 30 min.
(14) Count the number of positive clones and calculate the titer. Titer (pfu) = number of positive clones on the plate X number of dilutions X 10, host culture plate alone and phage plate alone should be free of bacterial clones with ampicillin resistance.
4. Immunoblotting to detect the presentation of exogenous proteins
The phage generated by pDTSPLAY-B empty vector does not contain exogenous genes on the surface, but only an anchoring protein (encoded by gene Ⅲ) with 6 histidine tags and Myc tags, and the band of this protein in SDS-PAGE is at the position of 65 kDa (the actual Mr is 45 kDa). pDISPLAY-B vector with the insertion of the exogenous protein coding gene, the band mobility will be obviously slowed down. The rate of band migration slows down significantly after insertion of the exogenous protein coding gene into the pDISPLAY-B vector. Two SDS-PAGE gels were electrophoresed at the same time, one gel was stained with Caulophylline blue to observe the protein sample volume, and the other gel was transferred to the membrane for immunoblotting.
(1) Electrophoresis and membrane transfer
Prepare regular 8% SDS-PAGE gel, take 10 μl of phage samples, add 10 μl of 2X protein electrophoresis sample buffer, and heat at 100℃ for 5 min to denature. Prepare two identical samples for electrophoresis at the same time. After electrophoresis, one piece of gel was stained with Caulophylline blue for 15 min to 60 min, and the amount of protein sample was observed after decolorization. The other piece of gel was transferred to nitrocellulose membrane by electrotransfer method, and then proceeded to the next step of immunoblotting.
(2) Immunodetection
Routine operation, after blocking, primary antibody incubation, secondary antibody incubation, NBT/BCIP color development, the results were analyzed.
A positive band should appear at 65 kDa in the empty vector control sample, and a positive band should appear at a position larger than the control sample in the display phage sample containing the exogenous gene (the size of the band depends on the length of the inserted gene). The immunoblotting assay not only provides information on whether the exogenous protein is demonstrated, but also on the relative amount of protein demonstrated. Sometimes a degradation band of the displayed protein may be detected (degradation of the exogenous protein may occur during phage packaging). However, since immunoblotting does not reveal the function of the phage-displayed proteins, e.g., some proteins, although expressed, may no longer be RNA-binding due to folding errors; further testing of the RNA-binding activity of the displayed proteins is required.
5. Detection of RNA binding activity of phage display proteins
(1) Preparation of target RNA
① Insert the target RNA sequence into pGEM derivative vector through HindⅢ and PstⅠ, and then construct recombinant clone [the recombinant clone can be constructed by synthesizing the DNA sequence corresponding to the target RNA sequence and the complementary DNA sequence (about 70 nucleotides each), and then cloning the synthesized double-stranded DNA sequence into the vector after in vitro annealing]. Before transcription, the recombinant plasmid was digested with AvaⅠ enzyme, and the linearized plasmid had to be purified before it could be used as the transcription template. The target RNA is transcribed as described in the Mega ( short ) scrip kits (usually 8 μg of template DNA per 20 μl of reaction system).
② Transcribed RNA is purified by phenol:chloroform extraction and ethanol precipitation.
③ Prepare 5%~8% denaturing acrylamide gel and pre-electrophoresis at 275 V for 30 min.
③ Prepare 5%~8% denaturing acrylamide gel, pre-electrophoresis at 275 V for 30 min. ④ Dissolve RNA obtained by precipitation with 4 μl TE, add 4 μl formamide and 2 μl sample buffer, heat at 85°C for 5 min, sample, electrophoresis at 250 V for 1~2 h.
⑤ Stain the gel with 1X TBE containing 10 μg/ml ethidium bromide for 15 min; decolorize the gel with 1X TBE for 2 times, each time for 10 min.
(6) Cut the gel to obtain the target RNA bands, put the cut gel containing target RNA into a 0.5 ml tube with an eyelet at the bottom, and then put it into a 1.5 ml centrifuge tube, and centrifuge the gel for 2 min to break it.
(vii) Add equal volumes of TE and 0.2% SDS to the tube containing the gel and mix for 1 h or overnight at 4℃. The suspension is added to a sterile Sephadex G-50 column and centrifuged at 150 g for 2 min. The eluate is collected in a clean centrifuge tube.
⑧ Add 50 μl of TE/SDS to the Sephadex G-50 column, centrifuge again at 150 g for 2 min, and collect the eluate into the same centrifuge tube.
⑨ Phenol: extract once with chloroform and again with chloroform. Transfer the supernatant to a new centrifuge tube, add 1/10 volume plus 3 mol/L sodium acetate, 2.5 times the volume of ethanol precipitation, 70% ethanol to wash the salt, and finally dissolved in 50 μl TE.
⑩ 1:100 dilution of RNA solution, detect A260, calculate the RNA concentration (= 40 μg/ml X A260 ). At the same time, 40 pmol of RNA was taken for electrophoresis to detect the recovery of RNA and whether it was degraded or not.
(2) RNA binding to streptavidin paramagnetic beads
The binding capacity of Streptavidin Paramagnetic Beads is 2~5 pmol of annealed RNA per 10 μl of Streptavidin Paramagnetic Beads, which is equivalent to ≥1012 binding sites (according to the calculation of one binding site for each RNA), therefore, 10 μl of Streptavidin Paramagnetic Beads can be used for multiple times after the binding reaction.
It is advisable to check the annealing efficiency of the RNA to DNA oligonucleotides when performing the first RNA binding experiments with Streptavidin Paramagnetic Beads. This can be done by reacting a constant amount of radiolabeled RNA with varying amounts of DNA oligonucleotides and detecting it by non-denaturing gel electrophoresis. The electrophoretic mobility of RNA annealed with DNA oligonucleotides is significantly slower. The radiolabeled annealed RNA can also be used to determine the degree of binding of RNA to streptavidin paramagnetic beads.
① Take 5 pmol of RNA (1 μl) and 5 pmol of oligonucleotide (1.5 μl), mix well and heat at 85°C for 3 min.
② Leave the sample for 3 min and cool to room temperature.
③ Add 1x volume of 2X TENT binding buffer.
④ Take 10 μl of Streptavidin Paramagnetic Beads, wash twice with 1 times volume of 1 mol/L NaOH, 0.1 mol/L NaCl; then wash twice with 1 times volume of 0.1 mol/L NaCl.
⑤ Resuspend the beads in 1x volume of 1X TENT binding buffer.
⑥ Remove the buffer, add DNA/RNA double-stranded body (5 pmol) and 5 μl tRNA (40 μg), mix and leave at room temperature for 30 min.
(vii) Wash the beads three times with 1X TENT buffer to remove the bound RNA.
⑧ Resuspend with 10 μl of 1X TENT buffer.
(3) Demonstration of phage binding to Streptavidin cis beads
The number of phage particles can be known by titer assay, and then the phage sample is mixed with magnetic beads containing excess target RNA, and the number of specifically bound phage is obtained by eluting non-specific binding. The ratio of the number of bound phages to the initial number of phages is the binding rate. Add lacZ-labeled phage to the experimental system. On plates containing X-Gal, blue clones are formed from host bacteria transfected with lacZ-labeled phage. The number of blue clones before and after screening can be counted to obtain data on non-specific background binding. In addition, it is possible to set up an experimental group of magnetic beads with unbound RNA or irrelevant RNA as a control for background binding.
① Phage binding reaction mixture: add about 1X1010 colony generating units of prepared "display" phage, 40 μg tRNA, and 40 U RNasin to 1X TENT binding buffer in a total volume of 30 μl. A 5-fold excess of lacZ-labeled phage can also be added to the reaction mixture. phage. An additional portion of the reaction mixture can be prepared and the extra portion can be used to detect the number of phages in the initial reaction mixture.
② Remove the buffer from the RNA-conjugated paramagnetic beads, add the phage-conjugated reaction mixture, and gently shake the reaction for 30 min at room temperature.
(iii) Rapidly wash the beads twice with 1X TENT buffer and resuspend the beads with 100 μl of 1X TENT buffer.
④ Dilute the 100 μl sample 6 times 1:100 (5 μl→495 μl), take 100 μl of 10-8, 10-10 and 10-12 dilution, add 100 μl of host bacteria to each dilution, incubate at 37℃ for 30 min, and then coat LB plate (containing 100 μg/ml penicillin, 2% glucose and 40 μg/ml X-Gal). Controls of different dilutions of the phage binding reaction mixture [from step (1)] not reacted with paramagnetic beads were set up at the same time.
⑤ Incubate the plates at 37℃ overnight and count the clones the next day. After the binding screen, the ratio of blue clones to white clones should be significantly reduced, and this ratio will be significantly reduced by selective binding. In general, 2%~5% of the demonstrated phage can bind to the RNA-magnetic beads, and the background binding should be less than 0.005%. Therefore, the purity of the target phage can be increased by 3~4 orders of magnitude after one round of selective binding.
6. In vitro screening of RNA-binding proteins
Phage display technology is suitable for studying the interaction between the RNA-binding domains in RNA-BP and RNA. The molecular mechanism of protein-RNA interactions can be studied by mutating the RNA-binding domains of proteins (randomly or selectively mutating certain amino acids that may have active functional significance). There are two approaches that are often used in the construction of phage display libraries. One is to replace a portion of the RNA-BP coding gene with a synthetic oligonucleotide containing one or more randomly mutated regions, and the other is to use a portion of the random oligonucleotide as a primer for PCR amplification and then integrate the PCR product into the target gene. The choice of the mutation method influences the wide range of amino acids covered in the study. Due to the limitations of the bacterial transformants (preferably 1010 ), libraries of complexity greater than 1010 cannot be fully screened in a single experiment. This does not mean that libraries larger than 1010 cannot be used, it just means that when analyzing complex libraries, not all clones can be analyzed in a single screening process. For a completely randomized mutation library, there are 21 possibilities for each amino acid site (20 amino acids plus the stop codon), and if it is hoped that the library constructed will be representative of all mutation possibilities, then at most 7 amino acids can be randomly mutated. Therefore, the methods and strategies for constructing mutant libraries should be determined according to the actual research problem.
(1) Preparation of phage display libraries
① Transform the host bacteria with the constructed phage display library, and coat LB plates (containing 100 μg/ml ampicillin and 2% glucose) at a density of not more than 100000 clones per 25 cm X 25 cm plate. If the library capacity is too large, it is necessary to incubate for several hours in liquid phase in a culture solution containing 100 μg/ml ampicillin and 200 μg/ml methicillin. The disadvantage of liquid-phase incubation is that some clones with growth advantages will be amplified in larger proportions.
② Scrape the clones grown on the culture plate with 2X YT culture solution, add 16% glycerol and store at -80℃. Bacteria cultured in liquid phase are centrifuged and resuspended with a small amount of 2X YT, added with 16% glycerol and stored at -80°C. This is the first generation of library preservation solution. This is the first generation of library preservation solution, called R-0 (Round-0). The quality of the mutant library can be checked by sequencing several monoclonal or polyclonal samples from R-0.
③ Take appropriate amount of R-0 phage library glycerol preservation solution and culture it in 30 ml 2X YT (containing 100 μg/ml ampicillin and 2% glucose) culture medium until OD600 reaches 0.5, and then infect it with auxiliary phage in the same way as in the previous section 3 "Preparation of display phage".
(2) In vitro detection of phage display library binding to target RNAs
The choice of screening method depends on the complexity of the phage display library. If the screening is performed on a library with high complexity, many clones, and different affinities, it is preferable to use the previously described screening method of pre-binding an excess of RNA to magnetic beads. If the phage libraries under study exhibit proteins with RNA affinities similar to each other, a more rigorous screening method can be used to reduce the concentration of target RNA; phages are co-incubated with DNA-RNA double-stranded bodies before binding to magnetic beads. In addition to the selection of methods, attention should be paid to the setting of controls during the experiment to facilitate the analysis of results. Fresh host bacteria should be cultured every day to facilitate titer determination.
① On the first day, dilute the overnight cultured host bacteria to LB culture solution at 1:500, and incubate at 37°C for 6 h with 250 r/min shaking for titer assay. Add phage display library and RNA-DNA double stranded body to the binding buffer (prepare as before, anneal the RNA and DNA first, then dilute them to the appropriate concentration, such as the RNA concentration close to or lower than Kd ), and the binding buffer should have the appropriate salt concentration, 2 μg/μl tRNA and 2 U/μl RNasin.
② After the binding reaction has reached equilibrium, add the magnetic beads (pre-washing method as before) and slowly rotate the reaction for 30 min at room temperature.
③ Wash the beads twice with 1 ml of binding buffer, and resuspend the beads with 100 μl of binding buffer. Then resuspend the beads with 100 μl of binding buffer. Obtain R-1 (round-1) phage and store it at 4°C.
④ Dilute the R-1 phage sample continuously, take 100 μl of 10-4 dilution, 10-6 dilution and 10-8 dilution, add 100 μl of host bacteria to each sample, incubate at 37℃ for 30 min, and then smear the plate for titer detection. The titer of phage without binding screening was also determined for comparison.
⑤ On the second day, dilute the overnight culture of host bacteria to LB culture medium at 1:500, incubate at 250 r/min at 37°C for 6 h, and then use it for titer detection.
(6) Count the number of clones on the culture plate used for titer assay, and calculate the titer of phage and R-1 phage that have been screened for binding at the end of the screening process, as well as the proportion of phage binding during the screening process.
(vii) Infect the host bacteria diluted and amplified in step (5) with an appropriate amount of R-1 phage, overload the host bacteria to ensure phage infectivity (about 104 cells/ml for 6 h after 1:500 dilution), and incubate at 37℃ for 30 min.
⑧ Infected cells were spread on LB plates (containing 100 μg/ml ampicillin and 2% glucose) and incubated at 37°C overnight.
⑨ On the 5th day, collect the clones grown on the culture plate with 2X YT culture solution, add 15% glycerol to prepare R-1 phage library, and store at -80℃. This phage display library can be infected with auxiliary phage for the next round of protein display, RNA binding, infection of host bacteria and analysis of screening results.
7. Analysis of res