A bacterial anti-transcription termination detection system for studying peptide-RNA interactions by screening recombinant cDNA libraries. This experiment was derived from the "RNA Laboratory Guidebook", edited by Xiaofei Zheng.
Operation method
Inhibition of bacterial transcription termination assays for screening RNA-binding protein experiments
Principle
A bacterial anti-transcription termination detection system for studying peptide-RNA interactions by screening recombinant cDNA libraries.
Materials and Instruments
N-expression plasmid E.coil N567 Receptor Bacteria Move -Materials and equipment For more product details, please visit Aladdin Scientific website.
Storage solution Buffer
Peptone medium Peptone plate N-expression plasmid combination library Culture plate
1. Detection of peptide-RNA interactions by comparing color changes in bacterial clones due to differences in β-galactosidase expression
(1) Stock solution: ampicillin (100 mg/ml), chloramphenicol (40 mg/ml, methanol solution), X-Gal (40 mg/ml, dimethylformamide solution), IPTG (100 mmol/L), which can be stored at -20℃ for several months.
(2) Peptone medium: 10 g peptone, 5 g NaCl, 1 L, sterilized and stored.
(3) Peptone plate: 10 g peptone, 5 g NaCl, 15 g agar powder, fixed to 1 L, autoclaved; after cooling, add 50 μg/ml ampicillin, 15 μg/ml chloramphenicol, 80 μg/ml X-Gal, 50 μmol/L IPTG.
(4) N-expression plasmid (pBR322-derived with ampicillin resistance), N-reporter gene expression plasmid ( pACYCJ84-derived with chloramphenicol resistance).
(5) E. coil N567 receptor bacteria containing N-expression plasmid and N-reporter plasmid (prepared by CaCl2 method), respectively.
2. Quantitative comparison of peptide-RNA interactions by β-galactosidase activity assay
(1) 储存液：1 mol/L 葡萄糖，1 mg/ml 维生素 B1，1 mol/L MgSO4 ，0.2 μm 滤膜过滤除菌；4 mg/ml 邻-硝基苯基-β-D-吡喃半乳糖苷（O nitrophrny-β-D-garactopyranoside，ONPG）溶解于 A 溶液，4℃ 保存；1 mol/L NaCO3, 0.1% SDS, and chloroform.
(2) A solution: 1.5 g K3HPO4, 4.5 g KH2PO4, 1.0 g ( NH4)2SO4, 0.5 g sodium citrate 2H2O, add water to 1 L, autoclaved and stored.
(3) Z buffer: 0.06 mol/L Na2HPO4, 0.01 mol/L NaH2PO4, 0.01 mol/L KCl, 0.001 mol/L MgSO4, 0.05 mol/L β-mercaptoethanol.
3 Preparation of recombinant libraries
(1) Stock solution: 200 mmol/L DTT ( -20℃ storage); 2.5 mmol/L dNTP mixture (-20℃ storage); 0.5 mol/L EDTA, pH 8.0; Phenol: Chloroform: Isoamyl alcohol (25:24:1 ) solution; 3 mol/L Sodium acetate ( pH 5.2); 50X TAE ( 242 g Tris base, 57.1 ml glacial acetic acid, 100 ml 0.5 mol/L EDTA, and water to 1 L).
(2) 20 μmol/L of a random DNA sequence library containing a condensed oligonucleotide with a fixed sequence at the end and NcoⅠ and BsmⅠ cleavage sites at the 5' and 3' ends, respectively.
(3) 20 μmol/L primer oligonucleotide with a fixed sequence complementary to the 3' end of the simple oligonucleotide.
(4) Enzyme and buffer: NcoⅠ(10 U/μl), BsmⅠ(5 U/μl), Sequencing enzyme 2.0(13 U/μl), T4 DNA ligase(1 U/μl).
4. Screening of new RNA-binding polypeptides from random recombinant libraries
(1) N-expression plasmid combinatorial library (N-cxpressor plasmid combinatorial library ).
(2) E.coli N567 sensory state (expression plasmid containing the reporter gene).
(3) Peptone medium, plate, ampicillin (100 mg/ml ), chloramphenicol (40 mg/ml ), methanol solution.
(4) Sterilized 96-well culture plate.
II. Methods of operation
1. Detect the interaction between peptide and RNA by comparing the color change of bacterial clones due to differences in β-galactosidase expression.
(1) Transform N567/pAC reporter bacteria with pBR N-expression plasmid: Take 10 ng (1~10 μl) of pBR plasmid and mix it with 50~100 μl of reporter bacteria in the sensory state, and then transfer it to ice bath for 10 min at 37°C for 2 min, and then add 0.5~1 ml peptone culture medium and shake it at 37°C for 1 hour.
(2) Take 1/5~1/10 of the transformation mixture and spread it on the culture plate containing antibiotics, X-Gal and IPTG, and incubate it at 34°C for 48 h. The mixture was then transferred to ice bath for 2 min at 37°C.
(3) The anti-transcription termination activity was detected by color comparison with positive/negative controls using a semi-quantitative scoring method: negative control strains containing the bison type nut reporter plasmid without the N-expression plasmid were scored as 0; positive control strains containing both the wild-type nut reporter plasmid and the N-expression plasmid were scored as + + + + + + + + + + + + + + + + + + + + + + + + + +.
2. Quantification of peptide RNA interactions by β-galactosidase activity assay
(1) Pick a single blue bacterial clone and incubate it overnight at 37°C in peptone culture medium containing the appropriate antibiotic.
(2) Dilute 50 times the overnight culture to 3~5 ml of culture medium (containing 22.4 mmol/L glucose, 1 μg/ml vitamin B1, 1 mmol/L MgSO4 and antibiotics) and incubate at 37 ℃ with shaking until the OD600 is about 0.2 (3~6 h). Add IPTG at a final concentration of 0.5 mmol/L and continue incubation until the OD600 reached 0.4~0.5, then measure and record the OD600 value.
(3) Mix 0.1 ml of bacterial culture with 0.9 ml of Z buffer, add 25 μl of chloroform and 12 μl of 0.1% SDS, and shake vigorously for 10 s to mix, at which time the solution should be mixed. 10~15 s after the addition of the ONPG solution and incubate at 28°C.
(4) When the solution turned yellow, the reaction was terminated by adding 0.4 ml of 1 mol/L NaCO3. The time from the addition of ONPG to the termination of the reaction was recorded.
(5) Centrifuge at 11000 g for 1 min to remove the precipitate, read the OD420 and OD550 values, and calculate the β-galactosidase activity using the formula: enzyme activity = 1000X( OD420-1. 6XOD550 )/(tX0. 1XOD600 ), t = reaction time.
3. Preparation of recombinant library
(1) Take 20 μl of oligonucleotide primer, 15 μl of simple oligonucleotide, 100 μl of sequencing enzyme buffer and 272.3 μl of water and mix well. Heat to 65℃ and then slowly cool to room temperature to anneal the primer and oligonucleotide.
(2) Add 25 μl of DTT, 60 μl of dNTP mixture, 7.7 μl (100 U) of sequencing enzyme and incubate at 37 ℃ for 20 min. 4 μl of EDTA was added to terminate the reaction. （The reaction was terminated by adding 4 μl EDTA. (A small amount of sample was retained prior to the addition of the sequencing enzyme to allow comparison of the extension products by 4% agarose gel electrophoresis.)
(3) Add 500 μl of phenol: chloroform: isoamyl alcohol solution, mix vigorously, centrifuge at 11,000 g for 1 min, and transfer the upper aqueous phase to a clean centrifuge tube; add 500 μl of chloroform, mix vigorously, centrifuge at 11,000 g for 1 min, and transfer the upper aqueous phase to a clean centrifuge tube. Precipitate the DNA with 1/10 volume of 3 mol/L sodium acetate and 2.5 times the volume of ethanol.
(4) Dissolve DNA with 290 μl of water, 50 μl of NEB buffer 2 and 40 μl of DTT (a small amount of sample was retained for comparison with the digested product by 4% agarose gel electrophoresis). Add 40 μl NcoⅠ and react at 37°C for 2 h (leave a small sample); add 80 μl BsmⅠ and react at 65°C for 2 h (leave a small sample). Phenol extraction and ethanol precipitation were performed as before, and the degree of cleavage was detected by 4% agarose gel electrophoresis.
(5) Separate the digested DNA with 12%, 1.6 mm acrylamide gel, cut off the corresponding strips, elute the DNA with 300 mmol/L sodium acetate, and then precipitate with 2.5 times the volume of ethanol.
(6) For the ligation reaction of 20 μl volume, add 50 ng of NcoⅠand BsmⅠdigested plasmid, 5 ng of DNA to be inserted (obtained in the previous step), 1X ligation buffer, and 2 μl of T4 DNA ligase. The ligation mixture was used to transform strain N567. The inoculum density of transformed bacteria was kept at 2000~3000 clones per 100 mm diameter plate.
4. Screening for new RNA-binding polypeptides from random recombinant libraries
(1) Primary screening: Take the prepared recombinant library (200 μl of ligand product) and add 1.5 ml of receptor reporter cells to a 150 mm diameter plate containing antibiotics, X-Gal and IPTG. Blue clones were picked (even weak blue clones were picked) and resuspended in 96-well plates (100 μl of peptone culture solution with antibiotics per well). Bacteria were cultured overnight to plateau stage, collected together and purified by alkaline lysis coupled with agarose gel electrophoresis to obtain the pBR plasmid.
(2) Secondary screening (excluding false positives caused by spontaneous mutation of the reporter plasmid): the obtained positive plasmids were co-transformed into reporter cells, coated plate screening, and the positive clones were picked and inoculated into 3 ml of peptone culture medium containing benzylpenicillin, and then cultured overnight to the growth plateau, and the plasmids of each clone were extracted separately.
(3) Third screening (to exclude false positives caused by non-specific binding to the target RNA): the obtained positive plasmids were transformed into strains containing specific reporter plasmids and strains containing non-specific reporter plasmids, and clones that could activate reporter genes in both types of strains were excluded.
(4) Fourth screening (to exclude false positives generated by base mutations at sites other than random libraries on the expression plasmid): the positive clones obtained from the third screening were sequenced, and then the positive sequences were synthesized again according to the sequencing results, and recloned into the human pBR, and then subjected to the anti-transcription termination assay once more.