Team:BUCT-China/Notebook
Notebook
I.Experiment Record-----Synthesis of Myristoleic Acid
Experimental time: July 23-30, 2021Experimental staff: Siying Yang
Experimental content: Amplification bacteria each four tubes ready to extract plasmids: T-C and C
Steps:
1.Remove the Glycerin tubes for T-C and C in the refrigerator
2.Eight LB liquid media were added with 4ulcana antibiotics each
3.Each tube takes 10ul of bacteria in the glycerin tube, T-C and C each add 4 tubes
4.37 degrees C shake culture
Experimental time: August 3-5, 2021
Experimental staff: Siying Yang
Repeat to C to do double enzyme cutting verification, found that always can not cut down, give up this group, do TU plasmids.
Experimental time: August 6, 2021
Experimental staff: Siying Yang
Experimental content: Refetching T-C plasmids
Experimental time: August 10, 2021
Experimental staff: Siying Yang
Experimental content: T-C running glue, glue recycling
Experimental results: running glue results have problems, cut incomplete
Experimental time: August 11, 2021
Experimental staff: Siying Yang
The TC sequencing results are as follows, correct
Connecting T-C and U (operation see T4 connection operation above)
Experimental time: August 17, 2021
Experimental staff: Siying Yang
Convert and Coating
Experimental time: August 19, 2021
Experimental staff: Siying Yang
The board grows colonies,Pick the gusty pipe and refrigerate.
II. Experiment Record-----Hydroxylation and Polymerization of Myristoleic Acid
[1] Experiment time: Friday, July 16, 2021
Experimental staff: Jin Shuming, Wang Ting, Zhang Hanyi
Experimental content: Enlarging Fadl, P450 BM3 plasmid copy-transformation
1. To prepare plasmid solution, the company synthesized 4mg of FadL-pUC57-Mini and P450 BM3-pET28a plasmids, centrifuged at 120,000g for 2min, added 40μl of ddH2O, and centrifuged again with a small centrifuge.
2. For transformation, take 2 tubes of XL10 competent cells on an ice box, add 2μl of a plasmid respectively, place on ice for 30 minutes, then take out in a water bath at 42°C for 90 seconds, and place on ice for 3 minutes. Add 500μl of KmR-resistant LB medium (100ml of LB medium with 100μl of kanamycin) on the ultra-clean table, and then place it on a shaker for 1 hour.
3. Coat the plate, after transforming and incubating for 1h, take out the EP tube 4000g and centrifuge for 2min, spread two plates on the ultra-clean workbench, and place them in a constant temperature incubator at 37℃ for constant temperature culture (colonies will be visible after 10h, and colonies will be seen after 16-18h. Too mature cannot be picked).
4. Primer design, according to P450 BM3-pET28a upstream and downstream design primer company synthesis, plan to obtain a large number of target gene fragments by PCR for double digestion.
[2]
Experiment time: Sunday, July 17, 2021
Experimental staff: Jin Shuming, Wang Ting, Zhang Hanyi
Experimental content: Fadl, P450 BM3 plasmid copy-picking culture
1. Take the plate coated on July 17, Fadl has only one single colony on one plate, and P450 BM3 has many single colonies on the other plate. On a clean bench, pick one each with a pipette tip and add them to two tubes of 5ml LB medium with 50μl kanamycin.
2. Incubate in a shaker at 37°C for 12 hours.
3. Take out the test tube, add 500μl of bacteria solution to 500μl of 50% glycerin aqueous solution, and store the bacteria in the refrigerator at -80℃.
[3]
Experiment time: Tuesday, July 18, 2021
Experimental staff: Jin Shuming, Wang Ting, Zhang Hanyi
Experimental content: Enlarging Fadl and P450 BM3 plasmid copies-inoculation culture
1. Take Fadl and P450 BM3 LB test tubes from the refrigerator, and add 100μl each to 4 tubes of 5ml LB medium (4μl kanamycin has been added) on a clean bench.
2. Place in a shaker overnight。
[4]
Experiment time: Wednesday, July 19, 2021
Experimental staff: Jin Shuming, Wang Ting, Zhang Hanyi
Experimental content: Fadl and P450 BM3 plasmid extraction 1. Take out the Fadl and P450 BM3 LB test tubes that were expanded on the 18th, transfer the bacterial solution to a 2ml EP tube and centrifuge at 12000rpm for 1min.
2. Discard the supernatant, add 250μl solution I (stored at 4°C), and resuspend the bacterial solution.
3. Add 250μl solution II, turn it upside down and rotate evenly, and let it stand for 2 minutes.
4. Add another 350μl solution III, and mix upside down immediately after adding.
5. Centrifuge at room temperature, 12000rpm, 10min.
6. Aspirate the supernatant and add it to the adsorption column, centrifuge at room temperature, 12000rpm, 1min.
7. Discard the filtrate, add 500μl Buffer HB to the adsorption column, centrifuge at 12000rpm for 1min, and wash the protein.
8. Discard the filtrate, add 700μl DNA wash Buffer to the adsorption column and centrifuge, 12000rpm, 1min, wash salt .
9. Repeat step 8 .
10. Centrifuge the empty adsorption column at 13000 rpm for 2 minutes to further dry the adsorption column.
11. The adsorption column is placed in the oven, and the lid is opened and dried for 5 minutes.
12. Take 30μl of EB touch the adsorption membrane with a pipette tip, add it to the adsorption column, leave it for 1-2min, and centrifuge at 12000rpm for 1min.
13. Add the eluate from the previous step to the adsorption membrane again in the same way, leave it for 1-2 minutes, and centrifuge at 12000 rpm for 1 minute.
14. At this time, the liquid at the bottom of the centrifuge tube is the extracted plasmid solution.
[5]
Experiment time: Monday, July 20, 2021
Experimental staff: Jin Shuming, Wang Ting, Zhang Hanyi, Shan Qi
Experimental content: P450 BM3 plasmid PCR running gel, Fadl double enzyme digestion and running gel.
1. Fadl double enzyme digestion and running gel and enzyme digestion system 50μl, 6 tubes in total
2. ddH2O 38.5μl, buffer 5μl, Fadl plasmid 3.5μl, NotI 1μl, NcoI 1μl, the order of addition is the same, calculate the amount of plasmid added according to the plasmid concentration, the amount of plasmid is required to be 100ng, and the concentration of Fadl plasmid is 330ng/μl.
3. Digestion at 37°C for 90 minutes
4. Glue 90ml TAE, 0.9g agarose.
5. Prepare new loading buffer: 100μl 6X loading buffer, 100μl ddH2O, 0.4μl gold view
6. Prepare a new maker: 100μl ddH2O, 20μl 6X loading buffer, mix well, aspirate 24μl solution and discard, then add 24μl maker to it.
7. The last maker to add samples is to take 4μl of the new maker and 10μl of the new loading and mix the solution.
8. For every 50μl system, add 20μl of Loading buffer and 14μl of Marker.
9. Running rubber 160v, 20mins .
10. Comparing with Maker after running the glue, double enzyme digestion is successful, and the glue is cut and recovered.
1) Fadl double enzyme digestion and gel recycling.
1. Under a long-wave UV lamp, use a clean blade to cut off the Fadl strips that need to be recycled. The smaller the size of the glue block, the better.
2. Put the cut gel into a 1.5ml centrifuge tube and weigh it.
3. Add the buffer solution Bingding Buffer according to the glue block: Bingding Buffer=1:1000.
4. Water bath at 56℃ for 10min until the colloid is completely dissolved, shake it every 2-3min to speed up the dissolution.
5. Add the solution obtained in the previous step to the adsorption column, leave it at room temperature for 1 min, centrifuge at 12000 rpm for 1 min, and discard the waste liquid in the collection tube.
6. After adding 300μl Bingbuffer, centrifuge at 12000rpm for 1min, and discard the waste liquid in the centrifuge tube.
7. After adding 700μl of WB buffer, centrifuge at 12000rpm for 1min, and discard the waste in the centrifuge tube.
8. Repeat step 7.
9. Put the adsorption column into a new 1.5ml centrifuge tube, add 15μl EB, leave it at room temperature for 1-2min, and centrifuge at 12000rpm for 2min.
10. Add the liquid obtained in the previous step to the adsorption column again, and centrifuge at 12000rpm for 1min.
11. The liquid collected at the bottom of the tube is the recovered Fadl after double digestion.
2) P450 BM3 plasmid PCR running gel
1. PCR system 50μl, 4 tubes.
2. ddH2O 17μl, 2X buffer 25μl, upstream primer 1μl, downstream primer 1μl, P450 BM3 plasmid 2μl, dNTP 1μl, Pfu enzyme 1μl. The order of sample addition is the same. Adjust the amount of water according to the amount of plasmid. The water is added first and not last.
3. Prepare gel with 45ml TAE and 0.45g agarose to prepare gel.
4. Add 4μl of gold view to the PCR product and then spot, add 6μl of 10000bp at the end maker.
5. Run rubber 160v, 20min. After running the gel, put the gel under the ultraviolet lamp to observe the band. After comparing with the maker, the band is normal, and proceed to the next step-cutting and recycling.
3) P450 BM3 cut rubber recycling
1. Under a long-wave UV lamp, use a clean blade to cut off the P450 BM3 strips that need to be recycled. The smaller the size of the glue block, the better.
2. Put the cut gel into a 1.5ml centrifuge tube and weigh it.
3. According to the glue block: Bingding Buffer=1: 1000, add the buffer Bingding Buffer.
4. 56℃ water bath for 10min until the colloid is completely dissolved, shake it every 2-3min to speed up the dissolution.
5. Add the solution obtained in the previous step to the adsorption column, leave it at room temperature for 1 min, centrifuge at 12000 rpm for 1 min, and discard the waste liquid in the collection tube.
6. After adding 300μl Bingbuffer, centrifuge at 12000rpm for 1min, and discard the waste liquid in the centrifuge tube.
7. After adding 700 μl of WB buffer, centrifuge at 12000 rpm for 1 min, and discard the waste liquid in the centrifuge tube.
8. Repeat step 7.
9. Put the adsorption column into a new 1.5ml centrifuge tube, add 30μl EB, leave it at room temperature for 1-2min, and centrifuge at 12000rpm for 2min.
10. Add the liquid obtained in the previous step to the adsorption column again, and centrifuge at 12000rpm for 1min.
11. The liquid collected at the bottom of the tube is the recovered P450 BM3 after double digestion.
III.Experiment Record-----The expression of hydroxylated collagen
Heat transduction (Transduction of plasmid into bacteria)
Experiment time:July 19, 2021Experimental staff: Shuming Jin, Xiao Xiao, Sijia Huang
pET28a-hCOL3 Kanamycin antibiotics
pET16b-L593 AmpR antibiotics
1. LB solid medium(0.5% 300ml sodium chloride. Yeast 0.5% peptone 1% agar 1.5%),In the high pressure steam sterilization pot 121 degrees Celsius, 30 min.
2. Take 50ul of competent cells BL21(DE3) melted in ice bath, add 1ul of target DNA, mix gently, and place in ice bath for 30min.
3. 42 degrees Celsius of water bath heat shock 90 seconds, and then quickly transferring pipe to the ice bath for 2 minutes, the process is not to shake the centrifugal tube.To join in each of the centrifuge tube 500 ul sterile SOC or LB culture medium, after blending at 37 degrees Celsius, 200 RPM cultivate 1 h.
4. Take 150 ul has transformed cells to the corresponding antibiotics LB AGAR culture medium, add 1/1000 of antibiotics. Put the plate culture medium into the shaker at 37 degrees Celsius and cultivate it overnight.
Induced expression of L593 and hCOL3
Experiment time:July 20, 2021Experimental staff: Jin Shuming, Yifan Zhu, Yunfeng Qu
1. The plump available bacteria were selected from the overnight plate culture medium and cultured in the liquid medium at 37 ℃ until OD600 reached 0.6. the expression of triglyceride was induced by adding 1 mM isopropyl β-D-glucosinolates. The cells were divided into 18 degrees Celsius and 30 degrees Celsius, and the blank group was cultured in the shaker for 8 hours.
2. Preservation of glycerol test tube: add a small amount of bacterial liquid to glycerin (- 80 ℃) to facilitate subsequent use.
Cell-breaking extraction of protein and Electrophoretic Verification of SDS protein
Experiment time:July 21, 2021Experimental staff: Shuming Jin, Xiao Xiao, Sijia Huang，Yunfeng Qu
1. 120 r/min, the centrifugal to the bottom of the precipitation, appeared a tube in qing dynasty, a total of 7 pipe, each join PBS(1*),
2. First blow wash in the centrifugal tube 500 multips to add 2.5 ml.After broken cell(Is better to clearing), Put in the ice water bath.
3. To break the cell machine use: open the machine power supply.Wipe the machine within the needles.Take appropriate to the size of the glass plate, add some ice cubes and water, the foam board，Water should not be foam board, will stay centrifuge tube into the broken cells.First place needles at the bottom of the centrifuge tube, but should not be in contact with the wall.Fixed. Adjust the centrifuge tube location make needle impending downward.First test, such as sharp sound and as can be.Formally began to break the cell.
3. After the cell breaking is completed, the liquid in the centrifuge tube is centrifuged and the precipitation is washed three times.
4. Sample preparation for electrophoresis: 50ul sample = 40ul (uniform precipitation and PBS) + 10ul (blue), boiling 10min
5. According to the description PAGE gel preparation: first select suitable thickness of the glass, this experiment choose 1.0 mm. Overlay the two pieces of glass in the correct way, clamping on the transparent carrier. According to the instruction manual configuration is required for the lower gel liquid, mixing, between pipetting gun in the glass. After filling the deionized water was added into the lower rubber surface level, the water filling. Place a period of time to clear water and gel layer.Pour water net. According to the instruction manual configuration suitable upper gel.The upper gel filled with can.Taking the thickness of the adapter insert top comb.Let stand until set.
6. Sample in the glue hole, Maker (two-color pre-dyed wide molecular weight protein) 5ul, nourishment 10ul, blank / control 5ul, start running glue, first 80v, set 30min (usually less than 30min) until the sample is pressed into a line, then set the current to 20mA, about 1h to the marginal bottom layer about 1.2cm voltage for electrophoresis
7. Decolorize the glue and observe the results.
Homologous recombination of L593 plasmid
Experiment time:July 25-26, 2021Experimental staff: Xiao Xiao, Yunfeng Qu
Improvement of the first round experiments: examine the L593 sequence and find that L593 could not be expressed normally due to the existence of this hydrophobic region (we guess it was a signal peptide): MKTVTIITIIVVIIVVILII MVLSKSCVMKTVTIITIIVVIIVVILIIMVLSKSCV. We decided to use homologous recombination to remove part of the sequence.
1. In the software snapgene, the primers used for homologous recombination are designed and sent to the company for synthesis. The newly bought primers are centrifuged (13000rpm 3.1*100ul for 2min) and slowly opened. After adding the sterilized distilled water (see instructions, 3.1*100ul), shake well, dissolve, and place the primers at room temperature.
2. PCR.
20ul system (9.3ul ddH20, 0.25ul upstream primers, 0.25ul downstream primers, 0.2ul template plasmid, 10ul dNTP, 1ul DNA polymerase).
PCR operation.
95 ℃ pre-denaturation for 30s.
95 ℃ denaturation for 15s.
62 ℃annealing for 15s.
72 ℃ extension (6327bp) 6min.
30 cycles.
72 ℃ complete extension of 5min
3. Homologous connection was carried out according to the instructions of the homologous kit and verified by DNA electrophoresis.
4. Heat transduction (Transduction of plasmid into bacteria）
pET16b-L593 AmpR antibiotics
[1] LB solid medium(0.5% 300ml sodium chloride. Yeast 0.5% peptone 1% agar 1.5%),In the high pressure steam sterilization pot 121 degrees Celsius, 30 min.
[2]Take 50ul of competent cells BL21(DE3) melted in ice bath, add 1ul of target DNA, mix gently, and place in ice bath for 30min.
[3]42 degrees Celsius of water bath heat shock 90 seconds, and then quickly transferring pipe to the ice bath for 2 minutes, the process is not to shake the centrifugal tube.To join in each of the centrifuge tube 500 ul sterile SOC or LB culture medium, after blending at 37 degrees Celsius, 200 RPM cultivate 1 h.
[4]Take 150 ul has transformed cells to the corresponding antibiotics LB AGAR culture medium, add 1/1000 of antibiotics. Put the plate culture medium into the shaker at 37 degrees Celsius and cultivate it overnight.
5. The fuller colonies were selected and sent to the company for sequencing to verify whether part of the sequence was successfully removed.
Synthesis of hydroxylated collagen by enzymatic modification in vitro
Experiment time:August 1-3, 2021Experimental staff: Shuming Jin, Sijia Huang, Yifan Zhu
1. The modified pET28a-hCOL3 and pET16b-L593 were expressed in E. coli BL21 (the specific steps have been written above)
2. 5 ug L593 prolyl hydroxylase were added to acceptor peptides at 0.5mg/ml in 50 mM Tris-HCl, pH 7.4, 100 μM FeSO4, 1 mM ascorbate, 100 μM DTT, 60 μM 2-oxoglutarate and 100 nCi of 2-oxo[14C]glutarate in a total volume of 100 μl and incubated at 37 °C for 45 min.
Hydroxylated Collagen purification
Experiment time:August 4-5, 2021Experimental staff: Shuming Jin, Yunfeng Qu, Xiao Xiao
1.Expand cultivating of hydroxylated Collagen: in vitro cultured species Numbers for: 30-228 - k species seeds, vaccination to 75 MLLB medium, quantity of 1%, 30 degrees and 18 degrees of inducing expression.
2.Enriching bacteria: 120x100r/min,1min, collecting Escherichia coli after induction and expression for 14h in 1min.
3.Heavy suspended bacteria: to use current now matches the PH = 7.2 Tris HCl buffer, heavy suspended bacteria 20 ml in 50 ml centrifuge tube. 4 s broken cell: set parameters, warming, cooling 6 s, broken cell 15 min, put in ice water.
4.Centrifugal, discard the clear liquid 120X100r/min
5.Protein that let stand for 10 min, the supernatant fluid with E1, 5 ml elution 3 times, E2, 5 ml elution 3 times, E3, 5 ml elution 4 times, discard 1 times E3 elution liquid, collecting liquid, 2, 3, 4 times marked 30-2;30-3;30-4(On behalf of 30 degrees).18-2;18-3;18-4 (on behalf of 18 degrees), a total of 6 pipe so that the follow-up measure protein content and electrophoresis detection.
6.E1:10mM imidazole，50mM Tris-HCl（PH=7.2），100mM NaCl
E2:30mM imidazole，50mM Tris-HCl（PH=7.2），100mM NaCl
E3:350mM imidazole，50mM Tris-HCl（PH=7.2），100mM NaCl
Measuring purified hydroxylated collagen concentration
Experiment time:August 6-7, 2021Experimental staff: Shuming Jin, Yunfeng Qu, Yifan Zhu
1. Using coomassie brilliant blue + bovine serum albumin (BSA) comparison method. (OD=596)
The preparation of standard solution:
2. Configuration of the sample under test.30-2;30-3;30-4; 18-2;18-3;18-4 each 10ul. Add 990 ul purified water, 5 ml1XG250, standard solution and sample under test are diluted 10 times after measuring the absorbance.
Standard solution diluted 10 times after the absorbance measurement results are as follows.
3. To make a figure as follows, absorbance and the concentration equation is: y = 0.0544 + 0.0011 x,
4. To measure the sample diluted 10 times absorbance measurement results are as follows, and turn it into the equations of the appeal of concentration.
5. The total elution concentration is 3 times the sum of elution concentration for their temperature.Due to the sample preparation when diluted 10 times diluted 10 times again, And three eluent volume of 15 ml, strain system for 75 ml, so the results are as follows:
Take the example of 30 degrees: 30, the total concentration was 0.003385+0.02514+0.006159+0.01209. Bacterial protein production quantity/(g/L):0.012058*15*10*10/75=0.241168. Conclusion: in terms of expressing protein content as an index, and the expression of 30 degrees condition is superior to 18 degrees.
IV.Experiment Record-----Cell culture
August 13
Extraction of primary muscle satellite cells from chickenAll according to the experimental design.
A few caveats:
1. When taking a muscle sample, try to select the muscle close to the bone.
2. We took a lot of muscle samples, so we carried them out in two times. The remaining part of muscle was soaked in DMEM-F12 medium and placed in an incubator. After 24 hours, the muscle tissue was taken out and cells were further extracted.
August 14
Cell extraction was observed & primary muscle satellite cells were extracted from the tissue of the previous day1. Observe the cells extracted yesterday
The red dots indicate muscle satellite cells and the green dots indicate tissue fragments, dead cells, and non-adherable red blood cells, which were cultured for 24 hours.
2. Continue to extract primary cells
This time, muscle tissue was digested in two ways: one was digested directly with collagenase, and the other continued to use a mixture of enzymes
August 15
Observation of muscle satellite cells1. The muscle satellite cells extracted on 13 August were presented
2.Cells extracted on August 14 were presented
Collagenase digestion: few muscle cells
Mixed enzyme solution digestion: As last time, the situation is optimistic.
For more detail of experiment of scaffold and cell culture, please go to the following links:
2021.igem.org/Team:BUCT-China/Design
2021.igem.org/Team:BUCT-China/Proof_Of_Concept
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