Team:HBUT-China/Notebook
We obtained the required nikABCDE fragment from the E. coli genome by PCR. The PCR system we use： ①.We used all the PCR products for gel electrophoresis. If the gel electrophoresis was successful, we would obtain the target fragment by gelation recovery. However, the result of gel electrophoresis did not show the band we need, which means our experiment failed. ①.We suspected that the temperature was too high during the PCR process, which caused the experiment’s failure, so we changed the conditions of the PCR and re-PCR the nikABCDE target fragment. But unfortunately, when we performed gel electrophoresis verification, we still did not get the strip we needed cause we we didn’t notice the time control for short strip. ①.We transformed the plasmid named PUC57-201801 into DH5α and coated DH5α on solid medium containing ampicillin resistance. ①.We designed PUC57-201802 and got the plasmid. ②.We transformed the plasmid into DH5α and coated DH5α on solid medium containing Ampicillin resistance. ①. After several experiments, we finally got the perfect temperature during the PCR process. We also obtained the target fragment nik ABCDE from E. coli by PCR. With the gel electrophoresis, we got the correct band , but other bands also showed up. We thought the low temperature caused mismatch during the PCR process. ②. We picked a single colony grown on the plate yesterday into a PA tube supplemented with 7 mL LB medium and 7 uL ampicillin. ①.Picked the monoclonal antibody into liquid LB. ①. We conducted temperature gradient experiments and set annealing temperature of 52.5 ℃, 53 ℃, 53.5 ℃, and 54 ℃, respectively to find the most suitable temperature . The final result showed a clear band at 52.5 ℃ and 54 ℃, but no band appeared at 53 ℃ and 53.5 ℃. We cut the gel of the target piece and performed the gelation recovery. ②. We tested the samples at 50 ℃, 52.5 ℃ and 54 ℃, but they showed no band. We thought the concentration of the plasmid after the gel-removal is too low,which results in the failure to be revealed by gel electrophoresis. ③.We preserved the cells containing the PUC57-201801 plasmid and took 5 mL bacterial solution to extract the plasmid. ④. The pSB1C3 linear plasmid and PC57-201801 plasmid were digested overnight at 37 ℃. The pSB1C3 digestion system we used: The PUC57-201801 digestion system we used: ①Conserve PUC57-201802 1. Use a pipette tip to take 500 µL "2018 02" into a single strain collection tube and mark "2018 02 Synthetic fragment". 2. Pipet 500 μL glycerol into the culture collection tube. 3. Place the strain collection tube in the -80℃ freezer. ②Extraction of plasmid and labeled "PUC57-201802 Plasmid". After extraction, the detection plasmid concentration was 424.7 ng/μL. ①. We set the annealing temperature at 52.5 °C and performed PCR on nikABCDE again, then took 1μL PCR product to detect its concentration, and verify the remaining products by gel electrophoresis. ②.Plastic recycling of pSB1C3 part and 201801 part. ③. The linear plasmids of pSB1C3 and PUC57-201801 were enzymatically ligated overnight at 15 °C. We labeled the plasmid after ligation with pSB1C3-01. The enzyme system we use: ①. Digest PUC57-201802 plasmid. System: 37 °C water bath for 12 hours. 2.PCR luxCDABE. 3.For electrophoresis, the running strip shows about 5.8 kb, indicating that the correct luxCDABE is obtained. ①.We converted all the enzyme products into DH5α and applied them to plates containing chloramphenicol resistance. ①. Ran gel electrophorsis to get the PUC57-201802 digestion product 201802 part. Result: Two bands appeared and one of them is 800 bp, which is the target band. ②. Plastic recycling of 201802 part. ③.Ligate pSB1C3 vector and 201802 part. System: pSB1C 32μL 201802 6μL Buffer 2μL T4DNA Ligase 1μL Double distilled water 9μL Add the whole system to the PCR tube, put it in the floating plate, and put it in the 15 °C incubator for 12h. ①.We picked 7 single colonies from yesterday's plates and added them to the PA tube containing 7 mL LB medium and 7 uL chloramphenicol. ①. We performed rapid detection of the plasmid of the 7 PA tubes. ②. No.4 tube and No.7 tube showed the best effect in the verification process, so we preserved the strains, extracted the plasmids, and took 4uL of the extracted plasmid for digestion to conform whether the plasmid was what we want. Enzyme-digestion verification system: ①.We verified the gel electrophoresis of yesterday's enzyme digestion system and obtained two bands with the fragment size of 200 bp and 2000 bp, which proved that the constructed PSB1C3 recombinant plasmid was successful. ①. Rapid detection of plasmid. Take 10 μL supernatant and 2018 02 plasmid for a total of 8 samples to run the gel electrophoresis for 25 min. Results: Compared with 201802 part, No.1 and No.3 had obvious leading bright bands, which were preserved and plasmids were labeled as “pSB1C3-02-1”, “pSB1C3-02-3”. ①.Digested the pSB1C3-01 circular plasmid with BamHI and Ran the gel electrophoresis. ②.We recovered the digested part and inserted nikABCDE into pSB1C3-01 by seamless cloning, and the mixed system was reacted at 37 ° C for 30 minutes. ③.We converted all of the products of the above seamless cloning system to DH5α. ④.Applied DH5α to the plasmid to a plate containing chloramphenicol resistance. Enzyme digestion system: Seamless cloning system: 1. Enzyme digestion verification 1. Enzyme digestion of “pSB1C3-02-1”, “pSB1C3-02-3”. System: plasmid 4μL Buffer Tango 1μL Xba I 0.3μL PstI 0.3μL Double distilled water 4.4μL 37 ° C water bath for 4 hours. Ran the gel electrophoresis: Results: The samples presented a band of 2044bp and 794 bp, verifying digestion successfully. pSB1C3-02: ②. Enzyme digestion The above two plasmids were digested by one enzyme. System: plasimd 20μL bufferTango 5μL stuI 3μL Double distilled water 22μL 37 ° C water bath, 8 hours ①.We picked 7 single colonies from yesterday's plates in PA tubes supplemented with 7 mL LB medium and 7 uL chloramphenicol. ①. Plastic recycling of luxCDABE. ②. Seamless connect luxCDABE and pSB1C3-02. System: pSB1C3-02 2μL luxCDABE 5μL Buffer 2μL Exnase II 1μL 37 ° C, 30 min, labeled "pSB1C3-02-luxCDABE". ③. Transformation of the ligated plasmid. Marked “pSB1C3-02-luxCDABE”. ①. We performed rapid detection of the plasmid in the 7 PA tubes. ②. We selected two tubes, preserved the strains and extracted the plasmids, then took 4 uL extracted plasmid for digestion to conform whether the plasmid was what we want. Enzyme digestion system: pSB1C3-01-nik 图: B:pSB1C3-luxCDABE: ①. Rapid detection of “pSB1C3-02-luxCDABE”. Result: “pSB1C3-02-luxCDABE”No. 3 and No. 5 may be right, so we extracted their plasmids. ②. We used XbaI and PstI to digest pSB1C3-02-luxCDABE, and ran the gel electrophoresis: It showed we get the pSB1C3-02-luxCDABE successfuly. ①.Plasmid digestion: System: plasmid 4μL Buffer Tango 1μL XhoI 0.3μL EcoRI 0.3μL Double distilled water 4.4μL 37 ° C water bath for 4 hours. Result: Both samples shows two bands, which aren’t line with our vision. After doing some research, we find out that there are two XhoI sites in PSB1C3 vector. Therefore in this emergent situation,we worked together and racked our minds,finally we figured out a perfect solution to this problem. Accoring to yesterday’s problem we changed our plan. 1. We simultaneously digested PUC57-201801 and pSB1C3-01-nik circular plasmid for 6 hours. 2. Plastic recycling of PUC57 vector、01-Nik part. 3. The PUC57-201801 linear plasmid and the nikABCDE fragment were ligated overnight at 15 °C and labeled pUC57-01-nik. pSB1C3-01-nik digestion system: PUC57-201801 digestion system: Enzyme system: ①.Plasmids digestion: Use EcoRI and PstI to digest PUC57-201801 and pSB1C3-01-luxCDABE. ②.Plastic recycling of PUC57 vector and 02-luxCDABE part. ③.Ligate PUC57 vector with 02-uxCDABE part. ①. The entire product was transformed into DH5α and coated on a plate containing ampicillin resistance. ②. We prepared a Tris-glycine SDS polyacrylamide gel electrophoresis separation gel to verify whether NikABCDE protein was inserted into pSB1C3-01 to make it PSB1C3-01-nikABCDE. Since the decolorization effect was not obvious, we chose to decolorize it overnight, but due to the volatilization of the decolorizing solution, the protein gel lost water and shrunk the next day. ①. We transformed the ligated plasmid(PUC57-01-Nik and PUC57-02-luxCDABE) into DH5α and coated DH5α on solid medium containing ampicillin resistance. ①. Picked a single colony from the plate into the PA tube supplemented with 7 mL LB medium and 7 uL chloramphenicol. ②. Re-ligated and converted the 201801 part and nikABCDE, and applied the linked product to a plate containing ampicillin resistance. ①. We picked 7 single colonies from yesterday's plates and added them to the PA tube containing 7 mL of LB medium and 7 uL of Ampicillin. ②.Religated PUC57 vector with 01-Nik part and 02-luxCDABE part seperately. ③.We transformed the ligated plasmid(PUC57-01-Nik and PUC57-02-luxCDABE) into DH5α and coated DH5α on solid medium containing ampicillin resistance. ①. We performed a rapid detection of the plasmid in the PA tube, but the final gel electrophoresis results did not show the plasmid we need. ②. Picked the single colony transformed yesterday to the PA tube, and we realized that the ampicillin resistance wasn’t durable. This time we strictly controlled the culture time. ①．Rapid detection of PUC57-01-Nik and PUC57-02-luxCDABE.But it didn’t show the strip we want. So we had to ligate them again. ②.We use StuⅠ to digest PUC57-201802. ①. Rapid detection of the plasmid in the PA tube, and the gel electrophoresis results showed the pUC57-01-nik plasmid we need. ②. Enzyme digestion verification: PUC57-Nik: ③. We preserved the strains showing the correct strip and extracted the plasmids. ①. Again we failed. ②. Plastic recycling of 02-luxCDABE part. ③. Ligated PUC57 vector with 02-luxCDABE part.(Unfortunately we failed) ④. Tested the chemiluminescence values,which can prove pSB1C3-luxCDABE works. 1.Tested the chemiluminescence values. ①. Ligated PUC57 vector with 02-luxCDABE part. ②. Ttransform the ligated plasmid(PUC57-01-Nik and PUC57-02-luxCDABE) into DH5α and coated DH5α on solid medium containing ampicillin resistance. ①. Picked 5 single colonies from yesterday's plates and added them to the PA tube containing 5 mL LB medium and 5 uL Ampicillin. ②. Rapid detection of PUC57-02-luxCDABE. ①. Ran the gel electrophoresis.And we find out that the five plasmids show the same strip,so we thought it might be right. ②. We used EcoRI and PstI to digest PUC57-02-luxCDABE. ①. Ran the gel electrophoresis and we get one strip,which means we succeed. ②. Plastic recycling of PUC57-02-luxCDABE part. ③. Enzyme digestion verification: pUC57-lux: ①. Ligated Nik part and PUC57-lux part. ①.Transformed the plasmid and performed spread plate. ②.After eight hours ,we found that there almost no singel colony.But we still picked three. ①.We found out that the three didn’t multiply,but some singel colonys showed up in the plate,so we picked 7.(Marked 1 to 7) ②.We performed the plasmid’s rapid detection.And found out number 2 had plasmid-strip,so we preserved the strain. ③.Cultured 50 uL strains in 5mL LB. ①.Extracted plasmid. ②.Digested the plasmid with EcoRⅠ and PstⅠ. ③.Performed gel electrophoresis and we found out that the strip was correct,so we did the plastic recycling. .Performed gel electrophoresis of PUC57-Nik-lux ,but it turned out to be incorrect.So we did the ligation again. ②.Transformed the strain PUC57-Nik-lux and performed spread plate. ①.Picked 7 singel colonys .(Marked 1 to 7) ②.We performed the plasmid’s rapid detection and found out number 5 had the fall-behind phenomenon. ③.Cultured 50 uL strains in 5mL LB. ①.Extracted plasmid and preserved the strain. ②.Digested PUC57-nikABCDE-luxCDABE with EcoRⅠ and PstⅠ and Performed gel electrophoresis. It showed two bands:one is 2.7kb the other is more than 10kb. ③.We did glue recovery of nik-lux part. ④.Ligated PSB1C3 part and nik-lux part. ①. Transformed pSB1C3-nik-lux and performed spread plate. ①. Rcovered the preserved pSB1C3-01-nik strain. ①.Picked 7 singel colonys .(Marked 1 to 7) ②.After eight hours we performed the plasmid’s rapid detection and found out that number 5 and 7 had plasmid-strip,so we extrcted plasmid. ③.Digested PSB1C3-Nik-lux with XbaⅠand PstⅠ for the enzyme digestion verification. ④.Tested the fluorescence intensity.But we didn’t test the no-load fluorescence intensity,so the result was useless. ①.Rcovered the preserved pSB1C3-01 strain and the activated pSB1C3-01-nik was placed into the refrigerator. ①. Number 7 showed three bands:7.6kb、4kb and 1.5kb.And it proved the ligation succeed and we got pSB1C3- nik-lux. ②.We took out the preserved strains:17iGEM、PSB1C3-02、PSB1C3-mRFP、PSB1C3-lux、PSB1C3-Nik-lux.And cultured 50 uL strains in 5mL LB separately. ①.Due to the protein gel instrument failure, we were unable to perform the experiment after preparing the Tris-glycine SDS polyacrylamide gel electrophoresis gel. Therefore, we had to re-cultivate the pSB1C3-01-nik strain and the pSB1C3-01 strain. ①.Added pSB1C3-nik-lux in LB with the concentration gradient of 500 50 5 0.5 0.05 0.005 0.0005 mM ,and tested the Optical Density(OD). The OD is :0.287、0.146、0.145、0.292、0.656、0.890、1.466 It supposed to be two situations: When C＜C1,OD stayed steady,when C＞C2,OD closed to 0. But it didn’t reflect the conclusion, so we kept cultruing the strains. ②.After 17 hours,we did the test again. It showed:0.02、0.034、0.031、1.056、1.047、1.078、1.134 When C＜0.5mM,it was stady,when C＞5,it closed to 0. Based on the above: We can say:we successfully got pSB1C3-nik-lux. ①. Prepared Tris-glycine SDS polyacrylamide gel electrophoresis separation gel. pSB1C3-01-nik and pSB1C3-01 were adjusted to the same initial Abs. By analyzing the result of the protein gel, we knew that pSB1C3-01-nik produced a darker color than pSB1C3-01 at the corresponding size, thus demonstrating the production of NikABCDE protein. ②.Pipette 1/1000 of pSB1C3-01-nik and pSB1C3-01 strains cultured yesterday to 800 μM of NiCl2 solution. The Ni2+ standard curve was detected. ①.We extracted the nickel ions in the dried bacteria with 0.1 mol/L HCl, and filtered.Finally detected the nickel ion concentration by detecting the Abs. ①.In order to expand the range of the nickel ion standard curve, we took the Ni2+ standard solution for the labeling experiment, but the Abs value was to high. We had to reduce the concentration of Ni2+ standard solution to prepare the calibration. ②.In order to determine the optimum Ni2+ concentration of E. coli, we cultured the bacteria in 0.5 nM and 0.8 nM Ni2+ medium in an inoculation amount of 100 uL. ①.We changed the concentration of Ni2+ standard solution several times to obtain a suitable range of Ni2+standard curve. The Ni2+ standard curve ②.For the next experiment, we cultured 100 mL of 3 bottles of PSB1C3-01 and PSB1C3-01-nik. ①. Add 0.5 nM Ni2+ solution to the 100 mL bacterial solution that had been cultured overnight, and the cells were cultured at the same Ni2+concentration. ①.The cells were collected and washed twice with double distilled water and EDTA, and they were disrupted by sonication. After disrupting the cells, Ni2+ in the cells was extracted by HCl, filtered through a filter membrane, and the filtrate was collected to detect Abs values: Method for extraction of plasmids: 1.Add 500 uL equilibration solution BL 12,000 rpm for 1 min to the adsorption column CP3 (put the column into the collection tube). Pour off the waste liquid from the collection tube and return the adsorption column to the collection tube. . 2. Take out the 5 mL overnight culture solution, add it to the centrifuge tube, then centrifuge at 12,000 rpm for 1 min, and try to aspirate the supernatant. Then collect the precipitate into a centrifuge tube. 3. Add 250 μL P1 to the centrifuge tube with the cell pellet (RNaseA has been added). Make sure the bacterial pellet is thoroughly suspended using a pipette or a vortex shaker. 4. Add 250 μL P2 to the centrifuge tube and gently invert it 6-8 times to fully lyse the cells. Mix them gently, don't violently oscillate, so as not to interrupt the genomic DNA, causing the extracted plasmid to be mixed with genomic DNA fragments. At this point, the bacterial solution should become clear and viscous, but the time should not exceed 5 min to avoid damaging the plasmid. 5. Add 350 μL P3 to the centrifuge tube, gently flip it up and down for 6-8 times, mix them well, and a white flocculent precipitate will appear. Centrifuge at 12,000 rpm for 10 min. 6. Transfer the supernatant collected in the previous step to the adsorption column CP3 (the adsorption column is placed in the collection tube), be sure not to aspirate the precipitation. Centrifuge at 12,000 rpm for 30-60 sec, drain the waste from the collection tube, and place the adsorption column CP3 into the collection tube. Method for plastic recycling : 1.Add 500 μL equilibrium liquid BL to the adsorption column CA2 (the adsorption column is placed in the collection tube）and centrifug at 12000 rpm for 1 min, the waste liquid in the collection tube is drained, and the adsorption column is returned to the collection tube. 2. Cut the 800 bp strip from the agarose gel and place it in a clean centrifuge tube and weigh it to 0.205 g 3. Add 205 μL accumulated solution PN to the gel block, and water bath at 50 ℃. During this time, gently press the centrifuge tube up and down to ensure that the rubber block is fully dissolved. 4. Add the solution obtained in the previous step to an adsorption column CA2 (adsorption column into the collection tube), place it at room temperature for 2 min, centrifuge at 12,000 rpm for 30-60 sec, pour off the waste liquid in the collection tube, and place the adsorption column CA2. Collect the tubes. 5. Add 600 μL rinse liquid PW (added absolute ethanol) to the adsorption column CA2, centrifuge at 12,000 rpm for 30-60 sec, drain the waste liquid from the collection tube, and put the adsorption column CA2 into the collection. Operation: 1. Add terminator, promoter, dd water to the PCR tube with enzymes. 2. Start PCR and set the parameters. (Extension time is three and a half minutes , annealing temperature is 50 ° C). 3. PCR finished. 1. Take the competent cell from the -80℃refrigerator and put it in the ice for 25 min. 2. All the plasmids that were ligated yesterday are added to the competent cell, then incubate for 10 min, heat shock at 42 °C for 90 s, and ice for 5 min. 3. Add 750 μL LB, shake at 45 ° C for 45 min, centrifuge at 10,000 rpm for 1 min. 4. Discard some of the supernatant,resuspend the rest solution, and apply to the resist plate contains Chl. 5. After the overnight culture, pick up monoclonal antibodies into 7 AP bottles with Chl resistance. Method for rapid detection of plasmids: 1. Add 500 μL from each of the 7 PA tubes to the 1.5 mL EP tube. 2. Centrifuge at 10,000 rpm for 1 minute, discard 450 μL the supernatant, and resuspend the remaining 50 μL the bacterial solution with a pipette tip. 3. Add 200 μL Tris and 200 μL chloroform to a 1.5 mL EP tube in the fume hood and mix them. 4. Add 50 μL the above mixed system to each of the 7 EP tubes containing the cells, and shake them for 1 minute with the vortexing machine. 5. Centrifuge at 12,000rpm for ten minutes, mix 10μL supernatant with 2μL loading buffer(6x), then run the gel electrophoresis. Standard measurement method: Take 0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 mL of Ni2+ standard solution (0.1g Ni2+ dissolve in (1+23) HCl and transfer it to 1L volumetric flask, add HCl to the scale, discard 1 mL and transferit to a 100 mL volumetric flask, add (1 + 23) HCl to the mark. Add 5 mL HCl to a 10 mL tube, and 0.5 mL of 500 g/L sodium potassium tartrate, 1.5 mL of 100 g/L NaOH and 0.5 mL of 160 g/L ammonium persulfate were added in order, let them stand for 3minutes. Further, 0.2 mL of 10 g/L dimethylglyoxime-ethanol was added to 10 mL of water, and let them stand for 15 minutes,then the absorbance was measured at 470 nm.
NoteBook
July 18
A:pSB1C3-nikABCDE:
July 19：
A:pSB1C3-nikABCDE:
July 20:
A:pSB1C3-nikABCDE:
July 22:
A:pSB1C3-nikABCDE:
B:pSB1C3-luxCDABE
July 23:
A:pSB1C3-nikABCDE:
B:pSB1C3-luxCDABE:
July 24:
A:pSB1C3-nikABCDE:
B:pSB1C3-luxCDABE:
July 25:
A:pSB1C3-nikABCDE:
B:pSB1C3-luxCDABE:
July 26
A:pSB1C3-nikABCDE:
B:pSB1C3-luxCDABE:
July 27:
A:pSB1C3-nikABCDE:
B:pSB1C3-luxCDABE:
July 28:
A:pSB1C3-nikABCDE:
July 29:
A:pSB1C3-nikABCDE:
B:pSB1C3-luxCDABE:
July 30:
A:pSB1C3-nikABCDE:
B:pSB1C3-luxCDABE:
July 31:
A:pSB1C3-nikABCDE:
B:pSB1C3-luxCDABE:
August 1:
A:pSB1C3-nikABCDE:
August 2:
B:pSB1C3-luxCDABE:
August 3:
A:pSB1C3-nikABCDE:
B:pSB1C3-luxCDABE:
August 4:
A:pSB1C3-nikABCDE:
B:pSB1C3-luxCDABE:
August 5:
A:pSB1C3-nikABCDE:
B:pSB1C3-luxCDABE:
August 6:
A:pSB1C3-nikABCDE:
B:pSB1C3-luxCDABE:
August 7:
A:pSB1C3-nikABCDE:
B:pSB1C3-luxCDABE:
August 10:
B:pSB1C3-luxCDABE:
August 11:
B:pSB1C3-luxCDABE:
August 12:
B:pSB1C3-luxCDABE:
August 13:
B:pSB1C3-luxCDABE:
August 14:
B:pSB1C3-luxCDABE:
C：pSB1C3-nikABCDE-luxCDABE:
August 15:
C：pSB1C3-nikABCDE-luxCDABE:
August 16:
C：pSB1C3-nikABCDE-luxCDABE:
August 17:
C：pSB1C3-nikABCDE-luxCDABE:
August 18:
C：pSB1C3-nikABCDE-luxCDABE:
August 19:
C：pSB1C3-nikABCDE-luxCDABE:
August 20:
C：pSB1C3-nikABCDE-luxCDABE:
August 21:
C：pSB1C3-nikABCDE-LuxCDABE:
A:pSB1C3-nikABCDE:
August 22:
C：pSB1C3-nikABCDE-luxCDABE:
A:pSB1C3-nikABCDE:
August 23:
C：pSB1C3-nikABCDE-luxCDABE:
August 24
C：pSB1C3-nikABCDE-luxCDABE:
A:pSB1C3-nikABCDE:
August 26:
A:pSB1C3-nikABCDE:
August 27:
A:pSB1C3-nikABCDE:
August 28:
A:pSB1C3-nikABCDE:
August 29:
A:pSB1C3-nikABCDE:
August 30:
A:pSB1C3-nikABCDE:
