Team:UESTC-China/Protocol
Bacteria
1.Overnight Cultivates
(1)Add 6mL autoclaved LB in a 12ml BD bottle.
(2)Pipet 6μL of 1000×antibiotic(100mg/mL) into the LB.
(3)Select a single colony using a sterile toothpick or pipette tip.
(4)Place toothpick or pipette tip in the culture tube and stir.
(5)Remove toothpick, or leave in the bottle.
(6)Place culture tube in incubator at 37℃ overnight shaking vigorously (220 rpm).
2.Plates Screening
(1)Pour 20mL autoclaved LB with appropriate antibiotic into each plate.
(2)Take the appropriate amount of bacterium suspension on the plate and spread it homogeneous.
(3)Seal the plate with parafilm, place upside down and culture at constant temperature of 37℃ for 12 – 14 hours.
3.Bacteria Preservation
(1)Mix 1ml with 1ml 40% glycerol.
(2)store at -20℃.
Molecular Biotechnology
We used different molecular biological methods in our project. All used methods are listed below.
1.PCR
In our project, in order to be able to finally amplify the target fragments. we try the following PCR systems and the procedure settings.
| Component | 50ul Reaction |
|---|---|
| I-5 Mix | 25ul |
| 10uM forward primer | 2ul |
| 10uM Reverse primer | 2ul |
| Temp | 1ul(2ng/ul) |
| ddH2O | 20ul |
| Segment | Cycles | Temperature | Time |
|---|---|---|---|
| Initial denaturation | 1 | 98℃ | 2min |
| Denaturation | 37 | 98℃ | 10s |
| Renaturation | 37 | 65℃ | 10s |
| Prolongation | 37 | 72℃ | 15s/kb |
| Terminal prolongation | 1 | 72℃ | 5min |
| heat preservation | 1 | 4℃ | Hold |
| Component | 25ul Reaction |
|---|---|
| 10uM Primer 1 | 1ul |
| 10uM Primer 2 | 1ul |
| Temp | 5ul |
| T3 enzyme | 18ul |
| Segment | Cycles | Temperature | Time |
|---|---|---|---|
| Initial denaturation | 1 | 98℃ | 2min |
| Denaturation | 33 | 98℃ | 10s |
| Renaturation | 33 | 57℃ | 10s |
| Prolongation | 33 | 72℃ | 20s/kb |
| Terminal prolongation | 1 | 72℃ | 2min |
| heat preservation | 1 | 4℃ | Hold |
2.Agarose Gel
(1)Weight 0.3g agarose, mix with 30ml 1x TAE in a conical flask (0.5g, 50ml for long dies).
(2)Heat the flask in the microwave oven until the solution becomes transparent, cool down to 40°C.
(3)Add 3ul GelRed into solution and mix evenly by shaking slightly (5ul GelRed for 50ml TAE)
(4)Pour the solution into a die and wait for the gel solidifying.
(1)Add 5ul(10ul) loading buffer into a 25ul(50ul) PCR product.
(2)Apply onto agarose gel together with makers.
(3)Run at 130V for 20 minutes for a full gel.
(1)Excise the agarose gel slice containing the DNA fragment of interest with a clean, sharp scalpel under ultraviolet illumination. Briefly place the excised gel slice on absorbent toweling to remove residual buffer. Transfer the gel slice to a piece or plastic wrap or a weighing boat. Mince the gel into small pieces and weigh. 100 mg of gel is equivalent to a 100 μl volume. Transfer the gel slice into a 1.5 ml microfuge tube.
(2)Add a 3x sample volume of Buffer DE-A.
(3)Resuspend the gel in Buffer DE-A by vortexing. Heat at 75°C until the gel is completely dissolved. Intermittent vortexing to accelerate gel solubilization.
(4)Add 0.5x Buffer DE-A volume of Buffer DE-B, mix. If the DNA fragment is less than 400 bp, supplement further with a 1x sample volume of isopropanol.
(5)Place a Miniprep column into a 2 ml microfuge tube. Transfer the solubilized agarosefrom Step 4 into the column. Centrifuge at 12,000xg for 1 minute.
(6)Discard the filtrate from the 2 ml microfuge tube. Return the Miniprep column to the 2 ml microfuge tube and add 500 μl of Buffer W1. Centrifuge at 12,000xg for 30 seconds.
(7)Discard the filtrate from the 2 ml microfuge tube. Return the Miniprep column to the 2 ml microfuge tube and add 700 μl of Buffer W2. Centrifuge at 12,000xg for 30 seconds.
(8)Discard the filtrate from the 2 ml microfuge tube. Place the Miniprep column back into the 2 ml microfuge tube. Add a second 700 μl aliquot of Buffer W2 and centrifuge at 12,000xg for 1 minute.
(9)Discard the filtrate from the 2 ml microfuge tube. Place the Miniprep column back into the 2 ml microfuge tube. Centrifuge at 12,000xg for 1 minute.
(10)Transfer the Miniprep column into a clean 1.5 ml microfuge tube (provided). To elute the DNA, add 25 μl of deionized water（65℃） to the center of the membrane. Let it stand for 1 minute at room temperature. Centrifuge at 12,000xg for 1 minute.
3. Ligation
| Component | 20ul Reaction |
|---|---|
| 10× Ligation buffer | 2ul |
| T4 Ligation enzyme | 1ul |
| Fragment of digestion | the moles rate of insert DNA to vector DNA is 5:1 to 10:1 |
| Vector of digestion | the moles rate of insert DNA to vector DNA is 5:1 to 10:1 |
| ddH2O | to 20ul |
Incubate at 22℃ for 2 hour
| Component | 20ul Reaction |
|---|---|
| 20ul Reaction | 15μl |
| Insert DNA | the moles rate of insert DNA to vector DNA is 5:1 to 10:1 |
| Vector | the moles rate of insert DNA to vector DNA is 5:1 to 10:1 |
| ddH2O | add to 20μl |
Incubate at 50℃ for 1 hour
4.Transformation
(1)Pipet 1ul-5ul ligation product into a 1.5 mL tube with 50uL competent E.coli Dh5α/ BL21(DE3)
(2)Put on ice for 30 minutes
(3)Incubate at 42°C for 45 seconds
(4)Put it back on ice for 5 minutes
(5)Add 500ul LB liquid media
(6)Incubate for 1 hour at 37℃ and 200 rpm
(7)Centrifuge tubes for 2 minutes at 6500 rpm
(8)Pipet 500uL supernatants out , pipet up and down the left liquid to suspend the cells
(9)Spread plate with glass bead
(10)Culture in an incubator overnight (14-18 hours) at 37℃
5.Confirmation
(1)Collect 5 ml of overnight LB culture. Centrifuge at 12,000×g for 1 minute to pellet the bacteria.
(2)Resuspend the bacterial pellet in 250 μl of Buffer S1 by vortexing.
(3)Add 250 μl of Buffer S2, and mix by gently inverting the tube for 7×.
(4)Add 350 μl of Buffer S3, and mix by gently inverting 6-8×. Centrifuge at 12,000×g for 10 minutes to clarify the lysate.
(5)Place a Miniprep column into an uncapped 2 ml Microfuge tube. Transfer the clarified supernatant from Step 4 into the Miniprep column. Transfer the Miniprep column and 2 ml Microfuge tube to microcentrifuge and spin at 12,000×g for 1 minute.
(6)Pipette 500 μl of Buffer W1 into each Miniprep column. Centrifuge at 12,000×g for 1 minute.
(7)Pipette 700 μl of Buffer W2 into each Miniprep column. Centrifuge at 12,000×g for 1 minute.
(8)Discard the filtrate from the 2 ml Microfuge tube. Place the Miniprep column back into the 2 ml Microfuge tube. Add 700 μl of Buffer W2 to the Miniprep column again and centrifuge at 12,000×g for 1 minute.
(9)Discard filtrate from the 2 ml Microfuge tube. Place the Miniprep column back into the 2 ml Microfuge tube. Centrifuge at 12,000×g for 1 minute.
(10)Transfer the Miniprep column into a clean 1.5 ml Microfuge tube. Add 50μl deionized water（65℃）to the center of the membrane. Let it stand for 1 min at room temperature. Centrifuge at 12,000×g for 1 minute.
In order to further validate the accuracy of the plasmid obtained from the positive clones selected by colony PCR, we select the double enzyme digestion for second verification.
| Component | 50ul Reaction |
|---|---|
| 10× FD Green buffer | 5μl |
| Forward enzymes | 1ul |
| Reverse enzymes | 1ul |
| Temp | 1ng |
| ddH2O | to 50ul |
(2).Put it in 37℃ Constant temperature incubator for 1h
(3).DNA Electrophoresis（No loading buffer）
Protein experiment
1.Protein concentration
(1) first clean the concentrated column with 20% ethanol, and then clean it with 20ml double distilled water.
(2) centrifuge 4000g for 30 minutes to remove the double distilled water from the concentrated column.Discard the waste liquid in the collection pipe.
(3) centrifuge 30mL bacterial solution at 8000rpm and 4℃ for 10min to obtain supernatant.
(4) collect the supernatant of 30ml in the concentrated column.Centrifuge 4000g frozen for about 30 minutes.Remove remaining eggs in the condensed column
White solution, stored in sterilized 1.5ml centrifuge tube.
2.SDS-PAGE
(1)Set the casting frames on the casting stands.
(2)Prepare the gel solution(as described above) in a separate small beaker.
(3)Swirl the solution gentlybut thoroughly.Pipet appropriate amount of separatinggel solution (listed above) into the gap between the glass plates.
(4)To make the top of the separating gel horizontal, fill in isopropanol into the gap until overflowing.
(5)Wait for 1h to let itgelate.
(1)Discard the isopropanol and you can see separating gel left.
(2)Pipet in stacking gel untill a overflow..
(3)Insert the well-forming comb without trapping air under the teeth. Wait for 40min to let it gelate.
(1)Make sure a complete gelation of the stacking gel and take out the comb.
(2)Take the glass plates out of the casting frame and set them in the cell buffer dam.
(3)Pour the running buffer (electrophoresis buffer) into the inner chamber and keep pouring
(4)after overflow untill the buffer surface reaches the required level in the outer chamber.
(1)Mix your samples with 5X lodaing buffer.(160L sample + 40L loading buffer).
(2)Heat them in boiling water for 5 min.
(1)Load protein marker in to the first lane.
(2)Load prepared samples into wells and make sure not to overflow.
(3)Then cover the top and connect the anodes.
(1)Set 80V before the downmost sign of protein marker reach the separating gel, it takes about 0.5h.
(2)Change the volt to 120V when the downmost sign of protein marker reach the separating gel, It takes about 0.5h.
(1)You can stop running when the downmost sign of the protein marker almost reaches the foot line of the glass plate.
(2)Take out the separating gel carefully, and dye in the coomassie brilliant blue solution(as described above) for 6h.
(3)Take out the separating gel and rinse it twice by UP water.
(4)Bleach it by destaining solution for 1.5h.
3. Protein content determination
(1) Dissolve BSA standard protein solution on ice.
(2) Prepare 50ul different concentrations of BSA solutions
| Protein concentration | 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 1mg/mlBSA(ul) | 0 | 5 | 10 | 15 | 20 | 25 | 30 | 35 | 40 | 45 | 50 |
| ddH2O(ul) | 50 | 45 | 40 | 35 | 30 | 25 | 20 | 15 | 10 | 5 | 0 |
(3) Measure the concentration of BSA protein OD595 with uv spectrophotometer
(4) Make standard curve with BSA protein concentration as the x-coordinate and OD595 as the y-coordinate.
(5) Add 1ml coomassie bright blue G250 reagent and 20ul sample into 1.5mlEP tube.
(6) Shake well and leave at room temperature for 5 minutes.
(7) Dilute the sample to be tested by five times.
(8) Take the diluted sample of 20 microns and 1ml kormas bright blue R250 in the EP tube, mix well and let stand 5 min
(9) Measure the absorbance value of the sample at 595nm with the microplate reader
(10) Calculate the corresponding protein concentration according to the standard curve.
4.IPTG Induction
(1) Pick colony from pET 21-L1-A2 transformants and inoculate 2mls of LB +100µg/ml amplicillin
(2) Grow overnight at 37℃
(3) Initially check OD600 of overnight at 230-250rpm: Should approximate to 3.0
(4) The next day, inoculate fresh 25ml aliquots of LB using 100µl of over night culture(s) (with OD600 approximating to 3.0) and grow for 2-3 hours at 230-250rpm until OD600 ~ 0.4 ( > 0.3 < 0.8)
(5) Check OD600 after 2 hrs: Generally speaking, OD600 should approximate to 0.4 if overnights at or about 3.0 under the aforementioned conditions
(6) Grow for about another 30min to attain OD600 0.4-0.6
(7) Place cultures on ice and cool shaking incubator to 20℃(~15-30 min)
(8) Now take 25 aliquots of microbial at OD600 ~0.4-0.6 and to one such aliquot add 250ul(1:100) of diluted IPTG(0.05M): This results in final conc. Of 0.5mM
(9) Induce at 16℃ for 16 hours
(10) Either make cell lysates straight away or place induced cultures at -20℃ pending lysate production
Detective methods
1.HPLC
(1).Flow velocity: 0.8 mL/min
(2).Column temperature: 25℃
(3).Mobile phase A: 0.025M phosphoric acid solution-acetonitrile(87:13;V:V)(modulated by triethylamine until pH=3.0±0.1)
Mobile phase B: Acetonitrile
| Timer(min) | Phase A(%) | Phase B(%) |
|---|---|---|
| 0 | 100 | 0 |
| 10 | 100 | 0 |
| 12 | 20 | 80 |
| 15 | 20 | 80 |
| 20 | 100 | 0 |
| 30 | 100 | 0 |
2.Green flurescence detection
(2).Take 800μl from yesterday's bacteria and prepare a 15ml system (100ml small conical flask), cultivate the bacteria to the logarithmic growth phase.
(3).separately add CIP to prepare a system having a CIP concentration of 0, 0.1, 1, 6, 10 μg/ml.
4.Detect OD600 and green fluorescence after 0h, 2h,4h,6h by a multi-function microplate reader.
•OD600 detection method:
in 96-well plate, 3 duplicate holes, minus LB blank
•Green fluorescence intensity detection method:
At each time point, 1.5ml of bacterial liquid was taken in a light-proof tube,centrifuged at 12000rpm for 20 minutes, washed once with distilled water, and resuspended once. Add 96-well plate 200, excitation wavelength at 475nm, emission wavelength.
3.Coupled enzymatic assay
(1).We incubated 2.0 mM CIP with 200ul cell disruption solution in a total volume of 1 ml of assay buffer (50 mM Tris [pH 7.6], 40 mM KCl, 10 mM MgCl2, 0.25 mg/ml NADH, 2.5 mM phosphoenolpyruvate, and 2.0 mM ATP) .
(2).8.0 μl pyruvate kinase and 4.0 ul lactate dehydrogenase were added .
(3).The mixtures were incubated at 37°C for 3 min.
(2).8.0 μl pyruvate kinase and 4.0 ul lactate dehydrogenase were added .
(3).The mixtures were incubated at 37°C for 3 min.