☼1.Transfer the ppET28A plasmid to competent cells. However, after the measurement, the concentration was found to be relatively low, so we decided to transform and extract the plasmid again.
☼2.The plasmid of tail bacteria was extracted but failed.
☼3.Prepare 500g/L glucose, fructose, sucrose and 200g/L xylose.
☼4.Transfer 50μL of marine bacteria from the sediment to TYS medium, and transfer 50μL of tail bacteria to LB medium.
☼1.Use 1ml of glycerol and 1ml of bacterial solution for bacteria preservation. (Bacteria: tail bacteria, JCM17730 and residential sediment)
☼2.Streak JCM17730 on the TYS plate without acetic acid; streak the sediment on the TYS medium without acetic acid.
☼3.Extract the tail plasmid.
☼4.Enzyme digestion of tail plasmid and pET28A (using BamHI and SacI), 37°C water bath for 4 hours.
☼5.After digestion, the gel is extracted.
Extract tail plasmid and pET28A plasmid.
☼1.Restriction digestion: pET28A plasmid first, then tail plasmid.
☼2.Gel Extraction：The pET28A plasmid and tail plasmid.
☼3.Connect pET28A and tail to construct pET-tail. Then transformed into bacteria JCM17730
☼4.Scribe the newly synthesized PET-tail.
☼5.Prepare LB and TYS medium.
☼1.pET-tail draws the plate.
☼2.pET-tail is transformed again.
☼1.Pick 3 marine bacteria for monoclonal culture and transfer to TYS vials. One bottle for bacteria storage; two bottles for colony PCR (16S) verification.
☼2.Transfer pMV-RS-tRNA (AMP resistant) to AMP resistant LB vial, store the bacteria, and extract the plasmid.
☼3.Pick 8 bacteria from the Kan-resistant PET-tail plate and put them in the LB vial. After extracting the plasmid, do restriction enzyme digestion and restriction enzyme digestion verification.
☼1.Re-extract the plasmids from 8 Kan-resistant pET-tail bacteria and perform restriction enzyme digestion and restriction enzyme digestion verification.
☼2.Re-check the colony PCR on 3 marine bacteria.
pET28A plasmid digestion (using BamHI, SacI); pKMV-tail plasmid digestion (using BamHI, SacI, ApaLI).
☼1.Add 1μL of CIP to the digested PET28A, dephosphorylate it, and take it out 30 minutes later.
☼2.Do pET28A and pKMV-tail gel extraction.
☼3.>Use 3μL PET28A, 1μL tail, 1μL T4 ligase, 1μL buffer and 4μL ddH2O to connect pET28A and tail.
☼4.Cut off the brightest electrophoresis band obtained from the gel extraction, and perform a second gel extraction.
☼5.Reconnect PET28A and tail (PCR at 22°C, 2h).
☼1.Colony PCR is as follows:
☼2.50μL of competent cells and 2μL of plasmid ice bath for 10min, heat shock, then incubate for half an hour, then spread the plate.
☼3.Pick 10 bacteria on the plate recovered by the secondary glue into the test tube, the test tube number is 1~10, and the bacteria 21~30.Put it on the shaker.
☼1.Extract the plasmid from the bacteria in 10 test tubes.
☼2.Pick colonies from two plates and do colony PCR verification as follows:
☼3.Do pET28A-tail enzyme digestion verification (10 plasmid ) (BamHI, SacI).
☼4.Extract the plasmid of pET28A.
☼5.Pick one bacterium from the pET28A plate into a test tube and place it on a shaker. Pick 2 of the 10 bacteria obtained in the test tube and put them in the LB vial, and then preserve the bacteria.
☼1.Preserve bacteria pET28A.
☼2.The first twopET28A-tail bacteria verified by restriction digestion is preserved.
☼3.Extract K1 and K2 plasmids.
☼4.Take 25μL-25mL BL21DE3 (pET28a-tail) into the LB vial for overnight culture, take 20-50mL transfer shake flask, culture for 2h, add IPTG induction, 6h later sample, protein electrophoresis.
☼5.pET28A-tail (Kan) (20μL competent cells, 2μL plasmid), pET28A-tail+pMV-RStRNA (Kan+Amp) (80μL competent cells, 8μL plasmid), put on a shaker. After one and a half hours, spread the plate, 12-16 hours later, pick a single clone to LB medium. After 12-14 hours, the bacteria are stored.
☼6.Prepare BoC-Lysine and IPTG solutions.
☼1.Pick the BL21DE3 (pET28a-tail) single clone to the LB vial and store the bacteria.
☼2.50μL of competent cells, 2.5μL of pET28a-tail, and 2.5μL of pMV-RS-tRNA (BL21DE3) were re-coated on a plate and cultured overnight.
☼3.Redo BL21DE3 competent cells.
☼4.5μL each of pET28a-tail and pMV-RS-tRNA, 100μL of competent cells, put them on a shaker after chemical transformation, take out one hour later and do plate coating.
☼5.Re-store 2 tubes of BL21DE3 (pET28a-tail) bacteria, take 25ml of bacteria liquid and transfer it to a 25ml LB vial, overnight, as the seed solution, transfer to a large shake flask the next day.
☼6.Transfer 4μL of BL21DE3 competent cells to a 4ml LB test tube.
Transfer 1ml BL21DE3 bacterial solution to an LB vial, and incubate on a shaker until the OD is around 0.8. After 2 hours of incubation, 500 μL IPTG was added to induce, and after 6 hours of incubation, samples were taken and protein electrophoresis was performed.
☼1.Transform plasmid pET28a-tail+pMV-RS-tRNA into WT, and spread the plate (amp+kan resistance) after 1 hour.
☼2.Transfer the pET28a-tail (BL21DE3) bacteria to the vial for culture.
☼1.Transfer yesterday's pET28a-tail (BL21DE3) bacteria (1% inoculation amount), and extract the plasmid. When the OD value is about 0.5, add the inducer IPTG (add to the final concentration of 0.02mM). Induction conditions: 22°C, 200rpm; later changed to induce conditions: 30°C, 200rpm.
☼2.Transform pMV-RS-tRNA to competent cells (amp); transform pET28a-tail+pMV-RS-tRNA (amp+kan) to competent cells.
☼3.The extracted plasmids pKMV-tail, pET28a-tail (JCM17730), pET28a-tail (BL21DE3), and pMV-RS-tRNA were detected by electrophoresis.
☼4.Re-transfer pMV-RS-tRNA bacteria and re-extract the plasmid.
☼5.Transform pMV-RS-tRNA into BL21DE3; transform pET28a-tail+pMV-RS-tRNA into BL21DE3.
☼6.Do single enzyme digestion verification as follows:
☼1.Transfer pET28a-tail (5μL) to BL21DE3 (50μL) again; transfer pET28a-tail (8μL) + pMV-RS-tRNA (1μL) to BL21DE3 (100μL); make pET28a-tail (BL21DE3) competent Cell, transfer pMV-RS-tRNA into it.
☼2.For cell-breaking pET28a-tail (BL21DE3) induced for 24 hours, the supernatant is required.
☼3.Transfer the mlrA-C1 strain to LB (kan), make 3 glycerol tubes tomorrow, and extract the remaining plasmids.
☼4.After sonicating the induced cells, there is a black precipitate, indicating that the power is too high, and protein electrophoresis will be done tomorrow.
☼1.Transfer two bacteria from the pET28a-tail (BL21DE3) on the plate, one for storing bacteria and one for competent cells.
☼2.Extract the pKMV-mlrA-C1 plasmid, do restriction enzyme digestion verification, the plasmid size is 3.2k, the verification is correct!
☼1.Make pMV-RS-tRNA competent, transfer pET28a-tail, and spread the plate.
☼2.The plasmid was extracted from JCM17730 (PCMCS3), digested with EcoRI-XhoI, and recovered after dephosphorylation for 0.5 hours. As a vector, it was about 4.7kb.
☼3.The plasmid of pMV-RS-tRNA was extracted and digested with EcoRI, XhoI and SacII to obtain 1.7kb+2.1kb, and 1.7kb was recovered.
☼4.Connect to construct pMCS3-RS-tRNA plasmid.
☼5.For transformation, double-transform the pET28a-tail + pMCS3-RS-tRNA plasmid into BL21DE3, pick a single clone, place it in an LB vial for culture, extract the plasmid and do enzyme digestion verification and colony PCR verification.
☼6.Transfer the bacteria JM109 (PCMCS3) to the LB vial, save the two tubes of bacteria first, and then extract the plasmid. After the plasmid is extracted, use EcoRI to verify.
☼7.After single enzyme digestion verification of PCMCS3 and RS-tRNA with EcoRI, electrophoresis was performed.
☼8.PCMCS3 and RS-tRNA were subjected to double and triple enzyme digestion verification.
☼1.The PCMCS3 was digested with EcoRI-XhoI for 6 hours, and CIP was added to dephosphorylate half an hour before the end of digestion, and the gel was extracted.
☼2.The pMV-RS-tRNA was digested with EcoRI-XhoI-SacII three enzymes, digested for 6 hours, and then the gel was extracted.
☼3.Protein electrophoresis after gel extraction.
☼1.JM109 (PCMCS3) and RS-tRNA were digested with double enzymes and three enzymes, respectively, and CIP was added to dephosphorylate half an hour before the end of digestion, and the gel was extracted. But it seems to have failed.
☼2.JM109 (PCMCS3) and RS-tRNA were digested with double enzymes and three enzymes, respectively, and CIP was added to dephosphorylate half an hour before the end of digestion, and the gel was recovered. But it seems to have failed.
☼3.After transformation of pMCS3-RS-tRNA, place it on a shaker.
☼1.Transfer bacteria 17287 to TYS, make a plate streak after turbidity.
☼2.Extract pMV-RS-tRNA plasmid and pET28a-tail plasmid.
☼3.Bacteria 17287 is used for streaking the plate and the other is for coating the plate.
☼1.Save two pMCS3-RS-tRNA bacteria, extract the plasmid, do EcoRI single enzyme digestion verification, the band size is 6.5 kb.
☼2.The first step of enzyme digestion verification failed, and the electrophoresis band size was 3.8kb.
☼3.Re-do pMCS3 and pMV-RS-tRNA digestion (4 hours), ligate (2 hours), and transform.
☼4.Transfer to pMCS3 and re-extract the vector plasmid.
☼5.Make the connection between pMCS3 and pMV-RS-tRNA, transform, and plate (amp resistance).
☼6.Transfer 100 μL of bacterial 17287 seed solution.
☼7.Transfer to JM109 (pMCS3), and extract the plasmid after 12-16 hours.
☼8.Transfer JCM17730, and shake the bottle up. Nonanoic acid, undecanoic acid, lauric acid and succinic acid are all 10g/L, and the dosage is as follows:
☼1.Extract the JM109 (pMCS3) plasmid.
☼2.Connect pMCS3-RS-tRNA, pick two bacteria on the plate, and transfer.
☼3.Pick 8 pMCS3-RS-tRNA bacteria into the test tube.
☼1.The pMCS3-RS-tRNA bacterial plasmid from yesterday was extracted and verified by restriction enzyme digestion, but the verification failed. And carry out colony PCR verification.
☼2.Test the standard sample with the kit.
☼3.Extract the pMV-RS-tRNA plasmid.
Use the kit to detect microcystin.
☼1.PMV-RS-tRNA was digested with three enzymes, and gel extraction was performed 6 hours later.
☼2.Gel electrophoresis was performed on JM109 (pMCS3) and pMV-RS-tRNA plasmids.
☼3.Retransform the bacteria of JM109 (pMCS3) and extract the plasmid.
☼1.After digestion of JM109 (pMCS3) and pMV-RS-tRNA, gel extraction and electrophoresis.
☼2.The colony PCR was run on gel and the solution was recovered.
☼2.The pMCS3 plasmid and pMV-RS-tRNA plasmid were digested with EcoRI-XhoI.
☼3.Transduct the pMV-RS-tRNA plasmid into trans-5α, plate coating, pick bacteria, and store bacteria.
☼4.Transfer P5-ψ1-dte-fucA-fucO-fucK bacteria
☼1.The pMCS3 plasmid and pMV-RS-tRNA plasmid that were digested yesterday were subjected to electrophoresis and gel extraction.
☼2.P5-ψ1-dte-fucA-fucO-fucK bacteria extract plasmid.
☼3.Test the sample with the kit.
☼4.Colony PCR verification.
☼5.Do P5-fucK three enzyme digestion, pET28a-tail two enzyme digestion, electrophoresis, gel cutting, gel extraction.
☼1.Pick colonies 4 and 6 from the LB1.7287 plate to culture in LB vials, and save the bacteria
☼2.Inoculate PMV-RstRNA bacteria into the test tube
☼3.Inoculate one of the PET28a-tail (BL21DE3) bacteria into the test tube and the other into the LB vial, one for storing bacteria and one for competent cells.
☼4.Transform PMCS3-RstRNA plasmid (5μl) into E. coli cell competent (50μl), and save the bacteria
☼5.Transfer PMCS3-RstRNA4 plasmid (5μl) to BL21DE3 (PET28a-tail) competent cells (50μl)
☼6.Transfer PET28a-tail (2.5μl) and PMCS3-RstRNA4 plasmid (2.5μl) to BL21DE3 (PET28a-tail) competent cells (50μl)
☼7.Send the original plasmid PMV-RstRNA (1), the bacteria 4, 5, PET28a-tail, and P5-CP1-dte-fuco-fuck found by PCR for sequencing
☼1.Save PMV-RstRNA1 two tubes of bacteria, each tube 500μl
☼2.Transfer test tube BL21 (PET28a-tail) as competent cells
☼3.Transfer the 20ml LB vial (PET28a-tail) BL21DE3
☼4.Pick up the monoclonal colonies on plate teas109 (PMCS3-RstRNA4) and BL21DE3 (PET28a-tail), put them in an LB vial, cultivate and store the bacteria
☼5.Perform three enzyme digestion and double digestion on P5-CP1-dte-fuco-fuck and PET28a-tail plasmids,
☼1.Recover P5:5.0kb and PET28a-tail:2.6kb by gel recovery, electrophoresis, inoculate 2μl and 6μl, respectively, for two hours, transform, transfer to the solution recovered PMV-RstRNA for enzyme digestion, and then gel recovery
☼2.Using the company's PMV-RstRNA as a template (1μl), use Q5 enzyme P, 100μl system, 5μl for verification, the electrophoresis band is 2kb, and the remaining 95μl is recovered from the solution
☼3.Enzyme digestion of PMV-RstRNA recovered from the previous solution (EcoRI, XhoI), gel recovery
☼1.Reconnect P5 and Taylor for two hours, then do conversion.
☼2.Re-transform the PMCS3-RstRNA (2μl:6μl) connected today, and put the rest in the refrigerator at 4℃.
☼3. Inoculate the five bacteria on the P5-tail plate painted yesterday into the test tube.
☼1.Transform the constructed plasmid into E. coli JCL16, spread the plate, pick the colonies into test tube culture, inoculate the bacteria into the shake flask, culture on the shaker, the duration is 9:18-12:43, and the OD600=0.8( Actual measurement 0.829) time. Add the inducer IPTG and continue to incubate until 15:24. At this time, the OD is about 3 (measured 5.1).
☼2.Centrifuge the bacterial solution, transfer it to a 24-well plate, and add the algal toxin.
☼3.Sampling for algal toxin detection (elisa kit).
Pick 8 colonies on the P5-tail plate made yesterday and culture them in a test tube
☼1.Connect the 9.25, 9.26, 9.26 P5-tail, P5-tail, PMCS3-RstRNA (2μl+6μl) and 3 new connections today made P5-tail (7μl+1μl), P5-tail (2μl+6μl) And PMCS3-RstRNA (4μl+4μl)
☼2.Use synthetic primers to remove 11-18 bacteria (1.8kb) picked out by P P5-tail
☼1.Validate the selection of monoclonal bacteria on the plates of 9.25 P5-tail, 9.28 P5-tail, 9.26 PMCS3-RstRNA, and 9.26 P5-tail
☼2.Correct PMCS3-RstRNA1-6 with primer P in the morning, pick it out and inoculate it into a vial, save the bacteria, extract and treat, and send it for sequencing
☼3.Pick the bacteria (345) on PMCS3-RstRNA into the LB vial, save the bacteria, and extract the plasmid
☼4.Save the bacteria 201501 picked by the teacher in the morning
☼5.Re-transfer PMCS3-RstRNA (4μl+4μl), P5-tail (2μl+6μl) to tran1-T1, put it in the shaker and spread the plate
☼1.Save the bacteria of PMCS3-RstRNA3/4/5 picked yesterday, extract plasmids from them, and send them for sequencing
☼2.Perform colony culture (2h) on the 4 monoclonal bacteria grown from the 9.28 P5-tail (7H), and then chemically transform PMCS3-RstRNA and PET28a-tail
☼3.The PMCS3-RstRNA (3, 4, 5) was subjected to restriction enzyme digestion verification, electrophoresis after 1 hour, no obvious results were obtained
☼4.Inoculate PET28a-tail (BL21DE3)
☼1.Re-transform PMCS3-RstRNA (4, 5) and PET28a-tail to BL21DE3
☼2.Competence of PET28a-tail (BL21DE3)
☼1.Raise 4 pKD46-Tu-XbaI.
⑴.plasmids Plasmid construction
⑵.Double-enzyme digestion of the PCR fragment and plasmid at the same time, and the product will be recovered by gel again.
⑶.Connect the obtained gene fragments and plasmid vectors, and transfer them to Escherichia coli competent.
☼2.Algal toxin detection
⑴.After the protein is induced with DNA, centrifuge it, take the bacteria, and reselect with 10ml lysis buffer.
⑶.Centrifuge, take the precipitate 20, reselect with lysis buffer; the supernatant is passed through the nickel column.
⑷.Perform protein electrophoresis on the purified protein, supernatant and precipitate, and check the bands according to the gel.
☼1.Protein electrophoresis separation gel, PET28a-tail, PET28a-tail+PMCS3-RstRNA, induced PET28a-tail for protein electrophoresis
☼2.PMCS3 gel recovery, PMV-RstRNA solution recovery and digestion gel recovery fragments (2μl+6μl)
☼3.Send the primers of P vector P5 and P fragment PET28a-tail to BGI
☼4.Convert 5μl, and put 5μl in refrigerator at 4℃
☼5.The first two uninduced PET28a-tail, PET28a-tail+PMCS3-RstRNA in protein electrophoresis, the last four are 15μl induced PET28a-tail, PET28a-tail+PMCS3-RstRNA, 10μl induced PET28a-tail, PET28a-tail+PMCS3-RstRNA
☼1.Transform the remaining 5μl of PMCS3-RstRNA connected yesterday
☼2.Make bacteria P for the bacteria grown on the PMCS3-RstRNA plate that was transformed yesterday, and the band size of the bacteria is correct (1.1kb) for two hours.
☼3.Pick 123 colonies on the plate into the LB vial
☼1.Extract and treat yesterday's 1, 2, and 3 bacteria and send them for sequencing
☼2.Re-inoculate bacteria 1, 2, and 3 into the test tube and extract the plasmid
☼3.Construct a 50μl PCR system for P5 and PET28a-tail (using Q5 mix)-P5F: 2.5μl, P5R: 2.5μl, template: 0.2μl, two Q5 mixes: 25μl, water: 19.8μl
☼4.P5 and PET28a-tail electrophoresis, P5 band is clear, tail band is not clear
☼5.Do two solution recycling, PET28a-tail glue recycling, and enzyme digestion glue recycling once (2.3kb)
☼1.1, 2, 3 plasmid concentration is 64, 70, 66 μg/ml
☼2.Perform electrophoresis on the P5 and tail recovered from the two solutions
☼3.Pst1 and Sat1 double enzyme digestion on PET28a-tail (recovered once from the solution), and glue recovery after 4 hours
☼4.Do the secondary solution recovery of P5 carrier
☼5.Do double enzyme digestion of PET28a-tail (Pst1, Sat1), do glue recovery, P5 concentration is 48μg/ml
☼6.Make the connection of P5-tail (P4: 2μl; tail: 6μl)
☼7.Inoculation bacteria: PET28a-tail (BL21DE3), PET28a-tail+PMCS3-RstRNA
☼1.Double enzyme digestion of PET28a-tail plasmid (Pst1, Sat1)
☼2.Storing bacteria on PET28a-tail+PMCS3-RstRNA
☼3.Make two transfers to PET28a-tail (one with IPTG induction, one without)
☼4.Do two inductions for PET28a-tail+PMCS3-RstRNA (one with IPTG and non-natural aa, one without)
☼5.Transform the connected P5-tail to trans109 and spread the plate
Inoculation bacteria PLMV-mlrA-c1,PLMV-mlrA-THN1,PET28a-tail+PMCS3-RstRNA
☼1.Configure 100×Boc-Lysine, 1000×IPTG
☼2.Transfer to PET28a-tail+PMCS3-RstRNA, 2 to 50ml large bottle, add 50μl antibiotics, 2ml bacterial solution, one at 30℃, one at 37℃, add IPTG and Boc-Lysine after 12h
☼3.Transfer PLMV-mlrA-c1, PLMV-mlrA-THN1 to 20ml vial, transfer JcM17730
☼4.Add IPTG (50μl) to PET28a-tail+PMCS3-RstRNA at 30℃ and 37℃, and induce Boc-Lysine (500μl) for 16 hours
☼5.Re-inoculation of bacteria PLMV-mlrA-c1 and PLMV-mlrA-THN1
☼6.Do JcM17730 antibiotic experiment
☼1.Wash the double plasmid at 30°C with 10ml PBS (0.05M) once, then centrifuge with 10ml and take the supernatant
☼2.Transfer the seed solution of PLMV-mlrA-c1 and PLMV-mlrA-THN1 to the 150ml system
☼1.PET-mlrA, PET-MBP-mlrA, add double distilled water, LB medium, harvest bacteria in the PET room, transfer to E. coli competent (Grains 5α), 6000rpm, 5min
☼3.Take 1ml of crude enzyme solution before centrifugation, take 1ml of supernatant after centrifugation, and elute the precipitate with 10ml lysis buffer
☼4.Pass the supernatant through the nickel column
☼5.Perform protein electrophoresis on the purified protein, supernatant and precipitate, and check the bands according to the gel
Stain: Coomassie brilliant blue, glacial acetic acid, water, methanol
Decolorizing liquid: glacial acetic acid, water, methanol