Difference between revisions of "Team:XMU-China/Experiments"

1. LB broth agar plates with 0.2% CMC are inoculated with the crude enzyme.
2. Then the plates are flooded with 1 mg/mL Congo Red solution for 1 h.
3. Congo Red solution is poured off into a toxic waste bottle and 1 M NaCl is added and leave the plates for another 1 h.
4. Then pour off NaCl solution.
5. Observe the phenomenon on the plates.

1. S. Lakhundi, Synthetic biology approach to cellulose degradation. (2012).
2. K. O. Duedu, C. E. French, Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass. Enzyme Microb Technol 93, 113-121 (2016).

● 4-Methylumbelliferyl β-D-cellobioside (MUC)
● Overnight culture grown in suitable medium
● Double distilled water (ddH2O)
● LB broth
● PBS buffer
● 1.5-mL centrifuge tube
● 50-mL centrifuge tube
● High speed refrigerated centrifuge
● Ultrasonic cell crusher
● Shaking incubator of 37 °C
● Black opaque 96-well plate (Costar^®)
● Microplate absorbance readers (TECAN^® infinite M200 PRO)

1. Dissolve MUC in ddH₂O to 4 mM as MUC stock (100×), Dilute with ddH₂O into working solution (5×).
2. Culture bacteria with target enzyme in suitable medium overnight.

1. Centrifuge at 5000 rpm in 4 °C for 10 min, collect supernatant and resuspend cells in PBS buffer.
2. Crush the cell suspension with ultrasonic cell crusher at power of 200 W for 66 times (3 seconds per time after gap of 6 seconds).
3. Centrifuge at 10000 rpm in 4 °C for 5 min, collect supernatant.
4. Add 200 μL MUC working solution (5×) into 800 μL culture supernatant or crushed cell supernatant as reaction system.
5. Add 200 μL MUC working solution (5×) into 800 μL LB broth or PBS buffer as background group.
6. Incubate under the condition of 37 °C, 200 rpm using a shaking incubator for reaction.
7. Take out one tube of reaction system into boiling water bath for 8 min to stop the reaction at the time of 15, 30, 60, 90, 120 minutes since reaction started.
8. Dilute reaction sample for 100 times and pipet 200 μL diluent into black opaque 96-well plate, measure fluorescence intensity (λ_ex = 364 nm, λ_em = 460 nm) with TECAN^® infinite M200 PRO.
9. Using fluorescence intensity to determine the activity of exoglucanase in samples.

1. S. Lakhundi, Synthetic biology approach to cellulose degradation. (2012).
2. K. O. Duedu, C. E. French, Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass. Enzyme Microb Technol 93, 113-121 (2016).

● PrimeSTAR^® Max DNA Polymerase (Takara^®)
● DNA template
● Primers (synthesized by Biosune^®)
● Nuclease-free 200-μL PCR tubes
● Biometra TAdvanced Thermal Cycler (Analyticjena^®)

1. Prepare reaction system:

2. Set PCR program:

● 1×PBS buffer
● Crude enzyme solution (store at 4 ℃)
● Glucose (Sangon Biotech^®)
● Cellobiose (Sangon Biotech^®)
● 5 mM sulfuric acid
● Double distilled water (ddH₂O)
● Agilent Technologies 1200 Series (Degasser, Quat Pump, ALS, TCC and RID)
● Agilent Technologies 1260 Inifinity (VWD)
● Column (Aminex HPX-87H Column, 300×7.8 mm)

● Sampling
1. Take 0.25 g of cellobiose in a 250-mL shake flask, and 40 mL of PBS buffer is added to each.
2. Each group was taken 0.8 mL of cellobiose solution as blank.
3. Add 0.8 mL PBS buffer to the shake flask and add 200 μL of PBS to the 1.5-mL centrifuge tube.
4. Add 10 mL crude enzyme solution to each of the experimental group and the control group, and mark the number.
5. Take 1 mL cellobiose solution that the crude enzyme solution is just added as blank. The shaker reaction is then carried out at 37 °C, 200 rpm.
6. Take 1 mL of the reaction mixture at specified intervals, mark the name, number and time period, and place the samples in a boiling water bath as soon as possible to quench the reaction.
Gradient I: 10, 20, 30, 40, 50, 60, 80, 100, 120, 150, 180, 240, 300, 360 min
Gradient II: 10, 20, 30, 40, 50, 60, 80, 100, 120, 150, 180, 240 min
Gradient III: 15, 30, 45, 60, 90, 120, 180 min
● Instrument pretreatment
1. Preparation of 5 mM sulfuric acid mobile phase
a) Add a certain amount of water to the beaker, add 535 μL 98% H₂SO₄ and transfer them to a volumetric flask. After cooling to room temperature, make up to 2 L.
b) Filter by vacuum pump and ultrasonic defoam for 30 min.
2. Preparation of glucose and cellobiose standard working curve samples (0.1, 0.2, 0.5, 1.0, 2.0, 5.0 g/L)
a) Prepare 5.0 g/L glucose and cellobiose solution.
b) Dilute to 0.1, 0.2, 0.5, 1.0, 2.0 g/L with other volumetric flasks based on a 5.0 g/L.
3. Instrument pre-cleaning
a) Loosen the pump valve and clean the outer passage to no air bubbles.
b) Tighten the pump valve. The flow rate was adjusted to 0.8 mL/min. After the baseline was substantially leveled, the flow rate was set to 0.5 mL/min and the pump was turned off.
c) Replace the mobile phase (pre-made 5 mM H₂SO₄) and start the pump.
d) Adjust the solution fill and open the pump valve. (counterclockwise)
e) RID—open heating valve—45 °C
f) RID—Control—Cleaning Reference Pool—Open—60 min
g) After the baseline is basically leveled, adjust the rectification speed to 0.2 mL/min and the upper pressure limit to 85 bar.
h) Install the column (Aminex HPX-87H Column, 300×7.8 mm).
i) Open the column oven at 65 °C, adjust the flow rate to 0.5 mL/min, and clean the reference cell for 1 h until the baseline is stable.
● Detection
1. Put in the wash bottle, sequence the parameters, set the path, name and operator, then fill in the sequence table.
2. Take a 1 mL of sample from the syringe and inject it into the sample tube through a 0.22-μm filter.
3. Place all samples in the sample tray according to the sequence table and feed them into the machine.
● Post-processing
1. After running the last sample, manually integrate and save the relevant data file.
2. Rinse for 1 hour and adjust the flow rate to 0.2 mL/min.
3. Close the column oven and turn off the pump and unload the column after the column temperature drops.
4. Replace the mobile phase (pure water), set the filling amount, and adjust the flow rate to 1 mL/mL.
5. RID—Control a cleaning reference pool for 30 min.
6. The flow rate is adjusted to 0.2 mL/min.
7. Turn off the pump (RID control - turn off the heater), software, liquid meter, and computer in sequence.

1．150 μL bacteria solution was inoculated into LB liquid mediums at 37 °C, 200 rpm until the OD₆₀₀ value of each reach to 1.
2．25 sterile tubes are needed for the following experiment. 250 μL bacteria solution from step 1 is inoculated into each tube with 3.75 mL LB liquid medium, and then cultured at 37 °C and 200 rpm.
3．Take out one tube every 2 hours, 3 mL bacterial solution is needed for optical density measurement at wavelengths of 600 nm, 577 nm and 458 nm.
4．Take out all of tubes when the OD₆₀₀ value exceeds 0.6. Then they are cultured at 37 °C and 200 rpm after addition of inducers (working concentration: lactose: 1mg/mL, arabinose: 1mg/mL IPTG: 1μg/mL).

● Culture dish
● Oxford cup with inner diameter of 6mm, outer diameter of 8mm and height of 10mm
● Chloramphenicol solution（1 mg/mL）
● LB liquid medium
● LB solid medium

1．Transfer plasmid pSB1C3 into E.coli DH5α and then inoculated it in LB liquid medium at 37 °C, 200 rpm until the OD₆₀₀ value of each culture reach to 1.
2．150 mL LB solid medium is heated for melt, and then cool it until the temperature decreases to 50 °C. Mix the medium with chloramphenicol solution (180 μL) and indicator bacteria solution (30 mL) and then pour them into the plate (about 12 mL medium per plate). Divide the plate into four areas on average after solidification.
3．First, place the Oxford cup vertically on the surface of the culture medium in the clean bench; then, gently pressurized to make it contact with the culture medium tightly; at last, add the corresponding samples(100 μL) into the Oxford cup.
4．Culture the samples at 37 °C for 16-18 hours, observe phenomena during the experiments and measure the diameter of inhibition zone.

● Culture and induction of bacteria
1. Add 200 μL glycerin bacteria into 10 mL LB liquid medium containing 50 mg/mL chloramphenicol. Incubate the culture in a shaker overnight at 37 °C, 200 rpm.
2. Add 1 mL of the culture above into 50 mL LB liquid medium containing 50 mg/mL chloramphenicol, and incubate it in a shaker of 37 °C and 200 rpm until OD₆₀₀ reaches 0.6.
3. Add IPTG to a final concentration of 500 μM to induce the expression of target genes.
4. Add the fluorescent dye 5(6)-carboxyfluorescein to a concentration of 10 mM.
5. Incubate the culture at 20 °C for 24 h.
● Preparation of wash buffer
1. Take 2.5 mL of 1 M Tris-HCl solution (pH 7.5) into a new 50-mL centrifuge tube.
2. Weigh 438 mg of analytically pure NaCl and pour it into the centrifuge tube above.
3. Add 50 mL of cold purified water. Adjust the pH to 7.5 with a pH meter. The wash buffer of 50 mM Tris-HCl, 150 mM NaCl, pH 7.5 is obtained.
● Preparation of samples and shooting
1. Pour the bacterial solution into a 50-mL centrifuge tube after induction. Centrifuge at 5000 rpm for 10 min to collect bacteria bodies.
2. Discard the supernatant, add 5 mL of wash buffer. Resuspend the pellets, and centrifuge at 5000 rpm for 10 min.
3. Repeat step 2. Wash the pellets until the color (orange) of the fluorescent dye 5(6)- carboxyfluorescein can not be seen. Discard the supernatant.
4. Add 25 mL of wash buffer and resuspend the cell pellets.
5. Take 20 μL of the resuspended droplets onto the coverslip. Gently attach the slide and coverslip together.
6. Apply a layer of clear nail polish around the coverslip to secure the coverslip and slide.
7. After the nail polish has dried, place it under the fluorescence microscope.
8. Select the oil microscope of the fluorescence microscope. The magnification is 100 times, shoot with bright field and BWA channel.
9. Save the bright field and fluorescent field photos with obvious effects.

1. J. Shin, et al., Display of membrane proteins on the heterologous caveolae carved by caveolin-1 in the Escherichia coli cytoplasm. Enzyme Microb Technol 79-80, 55–62 (2015).

● IPTG
● Pb(NO₃)₂
● Nitric acid
● LB liquid medium
● Chloramphenicol
● BL21 (DE3) glycerin bacteria to be tested
● Cryogenic refrigerated centrifuge
● ELAN ICP-DRC-qMS (PerkinElmer, SCIEX, Canada)
● 0.22-μm filter

● Preparation of samples to be tested
1. Add 200 μL glycerin bacteria into 10 mL LB liquid medium containing 50 μg/mL chloramphenicol. Incubate the culture in a shaker overnight at 37 °C, 200 rpm.
2. Add 1 mL the culture above into 50 mL LB liquid medium containing 50 μg/mL chloramphenicol, and incubate it in a shaker of 37 °C and 200 rpm until OD₆₀₀ reaches 0.5.
3. Add IPTG to a final concentration of 500 μM to induce the expression of target genes (DsbA-MBP, DsbA-PbBD and DsbA-MBP-CAV1).
4. Incubate the culture at 30 °C for 0.5 h.
5. Add Pb(NO₃)₂ to a final concentration of 50 μM. Shake the bacteria solution and divide them into four 50-mL centrifuge tubes evenly. Induce in a shaker at 37 °C, 200 rpm.
6. According to the set incubation time, periodically take out the centrifuge tubes from the shaker to stop the incubation.
7. Centrifuge at 5000 rpm for 10 min to collect bacterial bodies.
8. Discard the supernatant and wash the pellets with 1 mL of water. Repeat twice.
9. Resuspend the pellets in 1 mL of water and transfer the solution to a 1.5-mL centrifuge tube that has been weighed in advance.
10. Centrifuge at 5000 rpm for 5 min to collect bacterial bodies.
11. Discard the supernatant, dehydrate the bacteria at 60 °C for 12 h.
12. Weigh the tube and calculate the dry weight of the bacteria.
13. Add 1 mL of 2% dilute nitric acid to each 1.5-mL centrifuge tube. Digest bacteria in the water bath at 60 °C for 8 h avoiding light, and leave them at room temperature for 48 h to ensure digestion completely.
14. Filter the samples through a 0.22-μm filter after the digestion. The filtrates was diluted with purified water to obtain samples which can be tested.
● Determination of lead concentration in samples
1. The ²⁰⁸Pb element detection instrument is ELAN ICP-DRC-qMS (PerkinElmer, SCIEX, Canada).
2. Set mass spectrometry conditions: RF power, 1200 W; plasma gas flow, 15 L/min; auxiliary gas flow, 1.1 L/min; nebulizer gas flow, 0.88 L/min; lens voltage, 7.2 V; dwell time, 100 ms.
3. Set the injection mode as direct injection mode. Measure the signal intensity of ²⁰⁸Pb element in the samples, and substitute it into the corresponding range standard curve to calculate the lead concentration in the original samples. Obtain the Pb(Ⅱ) absorption amount per unit mass of bacteria.

1. https://static.igem.org/mediawiki/2010/0/08/Protocol_for_chemical_inducible_expression_of_MBP_%26_sample_preparation.pdf
2. C. Hui et al., Surface display of metal binding domain derived from PbrR on Escherichia coli specifically increases lead(II) adsorption. Biotechnol Lett 40, 837-845 (2018).

1. Sterilize the 5×M9 Salt Solution, 1 M CaCl₂ and 1 M MgSO₄ at 115 °C for 30 min.
2. Pass the 20% Glucose and 2% Cellobiose through a 0.22-μm syringe filter.
3. Mix the medium according to the following formula:

● LB Liquid Medium (Sangon Biotech^®)
● 1×PBS Buffer

● 0.2 M IPTG

● Shaking incubator of 37 °C & 20 °C
● Large centrifuge
● Spectrophotometer

1. LB liquid medium: Sterilization, at 115 °C, 30 min.
2. IPTG: removal of bacteria by 0.22-μm filter membrane.

● Cultivation
1. A single colony from LB broth agar plates stored in 40% glycerol at -80 °C is inoculated into 10 mL of LB liquid medium and grown overnight at shaking incubator of 37 °C.
2. Then inoculated at 1% by volume into fresh LB liquid medium.
3. Cell’s growth is monitored with a spectrophotometer equipped.
● Induced
1. When the value of OD₆₀₀ is between 0.6 and 0.8, added 0.1 mM IPTG to induce protein expression.
2. After induced under the condition of “20 °C, 200 rpm” for 24 h, the cells are harvested.
● Get the protein samples from the culture medium and the cells
1. Then the protein samples from the culture medium is obtained by centrifugation for 5 min, at 6500 rpm, 4 ℃.
2. The cell precipitation is washed twice with PBS buffer, and the final precipitation is resuspended in PBS buffer, which is equal to the volume of the original medium, and then the cells are broken on the ice. Then complete lysis on ice.
3. The supernatant is obtained by centrifugation for 5 min at 10000 rpm.

● Solarbio^® SDS-PAGE Gel preparation kit
● Bio-Rad^® Mini-PROTEAN System
● Double distilled water (ddH₂O)
● Glycine (produced by Solarbio^®)
● Tris Powder (produced by Solarbio^®)
● SDS (produced by Solarbio^®)
● Coomassie Blue R-250
● Isopropanol
● Glacial acetic acid
● Absolute ethyl alcohol
● Protein samples
● Prestained protein marker (produced by TransGen^®)
● 6×Protein loading buffer (produced by TransGen^®)
● 50-mL centrifuge tube
● Electrophoresis power supply
● Microwave oven

1. Wash the Bio-Rad^® Mini-PROTEAN System with ddH₂O.
2. Prepare samples for electrophoresis.

● Hand casting Polyacrylamide Gels
Please see SDS-PAGE with Silver Stain.
● Electrophoresis
Please see SDS-PAGE with Silver Stain.
● Coomassie Blue Stain
1. Prepare Coomassie Blue dyeing liquid and bleaching liquid:

2. Staining the gel:
a) Wash gels three times in ddH₂O.
b) Remove all water from staining container and add Coomassie blue dyeing liquid over the gel.
c) Heat with microwave oven in medium-high heat in 1 min.
d) Agitate in 40-60 rpm on a shaking table for 8 min.
3. Bleaching the staining:
a) Remove all water from staining container and add dyeing liquid back into container.
b) Wash gels three times in ddH₂O.
c) Add bleaching liquid over the gel.
d) Heat with microwave oven in medium-high heat in 1 min.
e) Agitate in 40-60 rpm on a shaking table for 20 min.
f) Pour the bleaching liquid back to container with activated carbon filtration.
g) Repeat step 3d-3f for 2 times.
4. Wash the gel with ddH₂O and image the gel.

● Guide to Polyacrylamide Gel Electrophoresis
Please see SDS-PAGE with Silver Stain.
● Usage of 6×Loading Buffer
Please see SDS-PAGE with Silver Stain.

● Solarbio^® SDS-PAGE Gel preparation kit
● Bio-Rad^® Mini-PROTEAN System
● Double distilled water (ddH₂O)
● Milli-Q Water
● Glycine (produced by Solarbio^®)
● Tris Powder (produced by Solarbio^®)
● SDS (produced by Solarbio^®)
● Beyotime^® Fast Silver Stain Kit
● Protein samples
● Prestained protein marker (produced by TransGen^®)
● 6×Protein loading buffer (produced by TransGen^®)
● 50-mL centrifuge tube
● Electrophoresis power supply
● Microwave oven

1. Wash the Bio-Rad^® Mini-PROTEAN System with ddH₂O.
2. Prepare samples for electrophoresis.

● Hand casting Polyacrylamide Gels
1. Wash the glass plate and dry with a mirror paper, assemble the glass cassette sandwich.
2. Prepare the resolving gel solutions without APS and TEMED, mix gently.

3. Add the APS and TEMED to resolving gel solution, pour 4.3 mL solution into the glass cassette sandwich using a pipet.
4. Using a pipet immediately overlay the monomer solution with ddH₂O.
5. Allow the gel to polymerize for 45-60 min, the gel is polymerized once you see a line from between the ddH₂O and the resolving gel. Pour off the overlay water.
6. Dry the area above the resolving gel with filter paper before pouring the stacking gel.
7. Prepare the stacking gel solutions without APS and TEMED, mix gently.
8. Add the APS and TEMED to stacking gel solution, pour the solution above the resolving gel using a pipet, pour until the liquid level reaches the upper edge.
9. Place the comb into the stacking gel and allow the gel to polymerize for 30-45 min.
10. Remove the comb by pulling it straight up slowly and gently, rinse the wells completely with ddH₂O.
Tips: Polymerized gel can be storage at 4 °C no more than 1 week when soaking in ddH₂O without removing the comb if the electrophoresis not be applied immediately after preparation of the gel.
● Electrophoresis
1. Prepare 2000 mL running buffer:

2. Prepare gels and assemble the electrophoresis cell:
a) Remove the comb form the gels and assemble the electrophoresis cell.
b) Fill the inner and outer buffer chambers with running buffer. Fill the upper buffer chamber of each core with 200 mL of running buffer; fill the lower buffer chamber to the indicator mark for 2 gels (550 mL) or 4 gels (800 mL) with running buffer.
3. Prepare samples
a) Add 6×Protein loading buffer into 5 times the volume of protein sample, mix gently.
b) Heat samples with boiled water bath for 8 min.
c) Load the appropriate volume of the sample on the gel (about 30 μL).
4. Connect the electrophoresis cell to the power supply and perform electrophoresis according to the following conditions:
a. Stage 1: 100V, 80 mA for 30 min
b. Stage 2: 150V, 120 mA for 40 min
5. After electrophoresis is complete, turn the power supply off and disconnect the electrical leads. Pop open the gel cassettes and remove the gel by floating it off the plate into water.
6. Stain and image the gel.
● Silver Stain
1. Wash the staining container and transform gel into staining container.
2. Fixation：
a) Prepare fixative liquid: 50 mL ethanol/10 mL acetic acid/40 mL Milli-Q water.
b) Pour fixative liquid over the gel.
c) Agitate in 60-70 rpm on a shaking table for 40 min.
3. Ethanol wash:
a) Discard fixation liquid, add 100 mL 30% ethanol over the gel.
b) Agitate in 60-70 rpm on a shaking table for 10 min.
4. Water Wash:
a) Discard 30% ethanol, add 200 mL Milli-Q water over the gel.
b) Agitate in 60-70 rpm on a shaking table for 10 min.
5. Sensitize:
a) Prepare sensitizer liquid: 99 mL Milli-Q water/1 mL sensitizer liquid (100×).
b) Discard Milli-Q water, add 100 mL sensitizer liquid over the gel.
c) Agitate in 60-70 rpm on a shaking table for 2 min.
6. Water Wash:
a) Discard sensitizer liquid, add 200 mL Milli-Q water over the gel.
b) Agitate in 60-70 rpm on a shaking table for 1 min.
c) Repeat water wash.
7. Silver stain:
a) Prepare silver reagent: 99 mL Milli-Q water/1 mL silver reagent (100×).
b) Discard Milli-Q water, add 100 mL silver reagent over the gel.
c) Agitate in 60-70 rpm on a shaking table for 10 min.
8. Water Wash:
a) Discard sensitizer liquid, add 100 mL Milli-Q water over the gel.
b) Agitate in 60-70 rpm on a shaking table for 1 min (No more than 1.5 min).
9. Developing:
a) Prepare developer reagent: 80 mL Milli-Q water/20 mL silver stain developer (5×)/0.05 mL accelerator.
b) Discard Milli-Q water, add 100 mL developer reagent over the gel.
c) Agitate in 60-70 rpm on a shaking table for about 2.5 min, until brown band appears.
10. Stop:
a) Prepare stop reagent: 95 mL Milli-Q water/20 mL silver stain stop reagent (20×).
b) Discard developer reagent, add 100 mL stop reagent over the gel.
c) Agitate in 60-70 rpm on a shaking table for 10 min.
11. Water Wash:
a) Discard stop reagent, add 100 mL Milli-Q water over the gel.
b) Agitate in 60-70 rpm on a shaking table for 4 min.
12. Wash the gel with ddH₂O and image the gel.

● Guide to Polyacrylamide Gel Electrophoresis
1. Bio-Rad. A Guide to Polyacrylamide Gel Electrophoresis and Detection. Bio-Rad Laboratories, Inc., 2019.
2. Green M R, Sambrook J. Molecular cloning. A Laboratory Manual 4th. (2012).
● Usage of 6×Loading Buffer
1. Manual of 6×Protein loading buffer:
http://www.transgen.com.cn/data/upload/pdf/DL101_2019-10-07.pdf
2. X. Chen, et al., Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system. Sci Rep 7, 44304 (2017).
3. S. Yang, et al., A HSP60-targeting peptide for cell apoptosis imaging. Oncogenesis 5, e201 (2016).
4. Y. Cheng, et al., Chicken DNA virus sensor DDX41 activates IFN-β signaling pathway dependent on chSTING. Dev Comp Immunol 76, 334-342 (2017).
● Silver Stain
1. Manual of Beyotime^® Fast Silver Stain Kit:
https://www.beyotime.com/Manual/P0017S%20%E5%BF%AB%E9%80%9F%E9%93%B6%E6%9F%93%E8%AF%95%E5%89%82%E7%9B%92.pdf
2. D. Xiang, et al., Expressions and purification of a mature form of recombinant human Chemerin in Escherichia coli. Protein Expression Purif 69, 153-158 (2010).