Team:Shanghai HS United/Experiment
Experiment
Plasmid 1—pMTL83151-J23100-Butyrate
1.Plasmid Extraction (AxyPrep Plasmid Miniprep Kit)
Collect 2 ml of bacteria culture in 2ml Eppendorf tube. Centrifuge at 12,000 rpm for 30 seconds, discard the supernatant.
Add 250 μl Buffer P1,resuspend the bacteria pellet by vortexing, until no sediment visible.
Add 250 μl Buffer P2, mix gently and reverse the Eppendorf tube 5-8 times.
Add 350 μl Buffer P3, mix by gently inverting the Eppendorf tube 5-8 times. Centrifuge at 12,000 rpm for 10 minutes.
Transfer the clarified supernatant from step 5 into a miniprep column, centrifuge at 12,000 rpm for 30 seconds. Discard the liquid in the collection tube.
Add 500 μl Buffer PD to miniprep column, centrifuge at 12,000 rpm for 30 seconds. Discard the liquid in the collection tube.
Add 600μl Buffer PW along the wall of the columns, centrifuge at 12,000 rpm for 30 seconds. Discard the liquid in the collection tube.
Repeat step 8.
Centrifuge at 12,000 rpm for 1 minute. Discard the liquid in the collection tube.
Open the lid of miniprep column for 1 minute to let the remaining liquid evaporate.
Transfer the miniprep column to a new 1.5 ml Eppendorf tube.
Add 50 μl double distilled water into the miniprep column (water should be preheated under 65 ℃), centrifuge at 12,000 rpm for 30 seconds.
Obtain its concentration using Microplate reader and note the value on the body of the tube. Place it in the -20˚C refrigerator.
2.KpnI restriction endonuclease digestion
Prepare a 0.2ml Eppendorf tube.
Prepare the reaction mixture: Add 5μl Buffer FD, 1μl KpnI solution, 2μl plasmid sample, and 42μl double distilled water, gently mixed.
Place the tube in the incubator, incubate at 37℃ for 1 hours.
3.Gel Electrophoresis
Gel preparation: apply 0.8g agarose, 80 ml TAE Buffer, and 8 μl nucleic acid dye into a 250 ml conical flask. Heat in microwave until no sediment visible.
Pour the gel into gel electrophoresis tank.
Sample preparation: apply 9μl loading buffer and 50μl production digested by KpnI into the sample wells. Mix 10μl marker(100-1000bp) with 2μl loading buffer to the leftmost well.
Electrophoresis for 20 minutes at 190V
Observe and record the bands under ultraviolet illumination.
4.Gel extraction
Prepare a 1.5ml Eppendorf tube.
Excise the fragments from the gel under ultraviolet illumination. Transfer it into the Eppendorf tube.
Add a 3× sample volume of Buffer DE-A, heat at 75℃ until no sediment visible.
Add a 0.5× sample volume of Buffer DE-B.mix.
Transfer the solution to a miniprep column.
Centrifuge at 12,000 rpm for 30 seconds, discard the liquid in collection tube.
Pipette 500 μl of Buffer W1 into the miniprep column, centrifuge at 12,000 rpm for 30 seconds. Discard the liquid in collection tube.
Pipette 700 μl of Buffer W2, centrifuge at 12,000 rpm for 30 seconds. Discard the liquid in collection tube.
Repeat step 8
Centrifuge at 12,000 rpm for 1 minute, discard the liquid in collection tube. Transfer the Miniprep column into a clean 1.5 ml Eppendorf tube.
Add 30 μl double distilled water. Centrifuge at 12,000 rpm for 1 minute.
5.Primer’s self-attachment to each other by their complementary bases.
Prepare a 0.2 ml Eppendorf tube
Prepare the reaction mixture: add 2μl T4 Buffer, 5 μl Primer J-23100/F, 5 μl Primer J-23100/R, 8 μl double distilled water.
Set the program for thermocycler: 95℃ 5 min, 95–85℃ at −2℃ /s; 85–25℃ at −0.1℃ /s; hold at 4℃.
Apply the program.
6.T4 DNA ligase
Prepare a 0.2 ml Eppendorf tube
Prepare the reaction mixture: add 2 μl of 10× ligation buffer, 50 ng of extracted DNA fragments from gel extraction, 4 μl of J-23100 from primer self-attachment mixture, 1μl T4 DNA ligase, and 13 μl double distilled water
Incubation at 16℃ for 12 hours.
7.DH5α competent cells transformation
Prepare a 1.5 ml Eppendorf tube. Transfer the T4 DNA ligase products in it, gently mix. Put the mixture on ice for 25 minutes.
Heat-shock at 42℃ for 1.5 minutes to let the plasmids absorbed by the competent cell.
Transfer it on ice for 2 minutes to let the cell membrane recovery.
Add 500μl LB culture
Incubate at 37℃ while shaking at 200-250rpm for 50 minutes.
Centrifuge at 8,000 rpm for 1 minute, resuspend the pellet with about 100 μl LB and spread the solid LB plate.
8.Colony PCR:
Prepare a 0.2ml Eppendorf tube.
Prepare the reaction mixture: add 10μl PCR Master Mix, 0.2μl J-23100-F, 0.2μl J-23100/R, 1μl transformed bacteria solution from the solid LB plate, and 8.6μl double distilled water.
9.Colony PCR procedure:
95℃ 5minutes → (95℃ 30seconds → 58℃ 30seconds → 72℃ 70seconds) ×25 cycle → 72℃ 5minutes → 4℃ ∞
Analysis the results by electrophoresis, detect the gel slice under ultraviolet illumination, select the ones that successfully transformed and send them for DNA sequencing.
Plasmid 2—p15A-J23200-lacY
Prepare a 0.2ml Eppendorf tube.
Prepare the reaction mixture: add 1μl template (plasmid with lacY gene), 10 μl Primestar mix, 0.4μl butyrate/F, 0.4 μl butyrate/R, 9.2 μl double distilled water
Set the program for PCR: 95℃ 5 minutes → (95℃ 30 seconds → 58℃ 30 seconds → 72℃ 20 seconds) ×30 cycles → 72℃ 5 minutes → 4℃ hold.
Run the PCR
2. PCR on p15A carrier.
Prepare a 0,2ml Eppendorf tube.
Prepare the reaction mixture: add 1μl template (p15A carrier), 10μl Primestar mix, 0.4μl J-23534, 0.4μl J-23535, 9.2μl double distilled water
Set the program for PCR: 95℃ 5 minutes → (95℃ 30 seconds → 58℃ 30 seconds → 72℃ 20 seconds) ×30 cycles → 72℃ 5 minutes → 4℃ hold.
Run the PCR
3. DpnI Demethylation modification
Prepare a 0.2ml Eppendorf tube.
Prepare the reaction mixture: add 1 μl DpnI, 5 μl Cutsmart buffer, 44 μl template (amplified plasmid 1)
React for 30 minutes under 37℃
MultiS recombination
preparation of 10µL system
Prepare the reaction mixture: template DNA, 2 µL 5×reaction buffer solution, 1 µL homologous recombinant enzyme
React at 37℃ for 30 minutes.
4. DH5α competent cells transformation
Prepare a 1.5 ml Eppendorf tube. Transfer the recombination products in it, gently mix. Put the mixture on ice for 25 minutes.
Heat-shock at 42℃ for 1.5 minutes to let the plasmids absorbed by the competent cell.
Transfer it on ice for 2 minutes to let the cell membrane recovery.
Add 500μl LB culture
Incubate at 37℃ while shaking at 200-250rpm for 50 minutes.
Centrifuge at 8,000 rpm for 1 minute, resuspend the pellet with about 100 μl LB and spread the solid LB plate.
Prepare a 0.2ml Eppendorf tube.
Prepare the reaction mixture: add 10μl PCR Master Mix, 0.2μl butyrate/F, 0.2μl butyrate/R, 1μl plasmid 3, and 8.6μl double distilled water.
Set the program of colony PCR: 95℃ 5minutes → (95℃ 30seconds → 58℃ 30seconds →72℃ 70seconds) ×25 cycle → 72℃ 5minutes → 4℃ ∞
Analysis the results by electrophoresis, detect the gel slice under ultraviolet illumination, select the ones that successfully transformed and send them for DNA sequencing. After sequencing, we transfer the successfully constructed plasmids into E coli Nissle 1917.
Plasmid 3—p15A-lacY-butyrate
1. PCR on plasmid 1
Prepare a 0.2 ml Eppendorf tube.
Prepare the reaction mixture: add 1 μl template (plasmid with butyric synthesis gene cluster), 10 μl Primestar mix, 0.4 μl butyrate/F, 0.4 μl butyrate/R, 9.2 μl double distilled water.
Set up the PCR procedure: 95℃ 5 minutes → (95℃ 30 seconds → 58℃ 30 seconds → 72℃ 20 seconds) ×30 cycles → 72℃ 5 minutes → 4℃ hold.
Run the PCR
2. PCR on plasmid 2
Prepare a 0.2 ml Eppendorf tube.
Prepare the reaction mixture: add 1 μl template (plasmid with lacY on p15A vector), 10 μl Primestar mix, 0.4 μl butyrate/F, 0.4 μl butyrate/R, 9.2 μl double distilled water
Set up the PCR procedure: 95℃ 5 minutes → (95℃ 30 seconds → 58℃ 30 seconds → 72℃ 20 seconds) ×30 cycles → 72℃ 5 minutes → 4℃ hold.
Run the PCR
3. DpnI Demethylation modification
Prepare a 0.2ml Eppendorf
Prepare the reaction mix: add 1 μl DpnI, 5 μl Cutsmart buffer, 44 μl template (amplification of plasmid 1)
React for 30 minutes at 37℃
4. MultiS recombination
Prepare a 0.2 ml Eppendorf tube
Prepare the reaction mixture: add PCR-recycling fragment1, vector-fragment2, 4 μl reaction buffer, 2 μl MultiS mix, and double distilled water up to 20 μl.
Incubate for 30 minutes at 37℃
Cool on ice for 5 minutes.
5. DH5α competent cells transformation
Prepare a 1.5 ml Eppendorf tube. Transfer the recombination products in it, gently mix. Put the mixture on ice for 25 minutes.
Heat-shock at 42℃ for 1.5 minutes to let the plasmids absorbed by the competent cell.
Transfer it on ice for 2 minutes to let the cell membrane recovery.
Add 500μl LB culture
Incubate at 37℃ while shaking at 200-250rpm for 50 minutes.
Centrifuge at 8,000 rpm for 1 minute, resuspend the pellet with about 100 μl LB and spread the solid LB plate.
6. Colony PCR
Prepare a 0.2 ml Eppendorf tube.
Prepare the reaction mixture: add 10 μl PCR Master Mix, 0.2 μl butyrate/F, 0.2 μl butyrate/R, 1 μl plasmid 4, and 8.6 μl double distilled water.
Set the program of colony PCR: 95℃ 5minutes → (95℃ 30seconds → 58℃ 30seconds →72℃ 70seconds) ×25 cycle → 72℃ 5minutes → 4℃ ∞
Analysis the results by electrophoresis, detect the gel slice under ultraviolet illumination, select the ones that successfully transformed and send them for DNA sequencing. After sequencing, we transfer the successfully constructed plasmids into E coli Nissle 1917.
Fermentation test
Prepare M-9 culture solution by adding:
100 mL of M-9 solution (Na2PO4.7H2O 1.28g, KH2PO4 0.3g, NaCl 0.05g, NH4Cl 0.1g)
1 ml of 1M MgSO4
10 ml of 10% glucose solution
Add 0.05 ml of 1M CaCl2
Add ddH2O up to 500 ml
Repeat steps 1-6 but change glucose to a mixture of glucose and lactose
Prepare 10 100 ml conical flasks, 5 for each kind of M-9
Add 2ml the bacteria culture into the conical flasks for fermentation
Take samples out of the beaker to measure the concentration of carbohydrates using OD600 at 0, 4, 6, 8, 24 hours
Analyze the results and see if there is a decrease in sugar concentration
Primer sequences
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