2 Molecular experiment
PCR(Polymerase chain reaction)
Introduction
This step is to amplify the target gene from the purchased plasmid by PCR reaction.
PCR enables the primers to bind specifically to the plasmid and amplify the genes of interest in several
thermal cycles.
Procedure
1. Add 25μL 2$\times$Phanta SE Buffer to each tube.
2. Add 2μl of forward primer
and reverse primer to each tube.
3. Add 1μL of template DNA to each tube.
4. Add ddH2O 19μL
to each tube.
5. Finally, add 1μL of Phanta SE Super-Fidelity DNA Polymerase to each tube.
6. Put the
tubes into the PCR Amplifier for PCR.
Thermocycling conditions for PCR
| Step |
Temperature |
Duration |
Cycles |
| Initial denaturation |
98℃ |
30s |
1 |
| Denaturation |
98℃ |
10s |
35 |
| Annealing |
(Tm+5)℃ |
5s |
| Extension |
72℃ |
5-10s/kb |
| Final extension |
72℃ |
1min |
1 |
| Hold |
4℃ |
∞ |
1 |
Agarose Gel Electrophoresis
Introduction
This process enables DNA fragments of different lengths moving through an agarose gel at different speeds to
cause them to separate. This step allows us to isolate and examine the target gene and test whether the
target gene has been successfully transferred into the recipient cell.
Procedure
Agarose Gel Preparation:
1. Add 0.3g agarose and 30 mL 1 x TAE to the conical bottle.
2. Heat the agarose
until it dissolves.
3. After the agarose solution has cooled down slightly, add 3μl of Ultra GelRed and
shake well.
4. Pour agarose gel into gel caster and install the comb into the gel caster.
5. Wait
until the agarose gel has solidified.
Sample Loading and Running:
1. Place agarose gel in the electrophoresis chamber.
2. Add 1 x TAE to the
electrophoresis chamber until it covers the agarose gel.
3. Load the sample and 10μL DNA marker to the
wells of agarose gel.
4. Set the voltage and running time of electrophoresis.
5. Start
electrophoresis.
6. After electrophoresis, remove the agarose gel from the horizontal
electrophoresis.
7. Put agarose gel under ultraviolet light, observe and take pictures.
8. Cut off the
agarose gel containing the desired gene.
DNA Extraction
Introduction
The procedure allows the DNA in the agarose gel to be purified.
Material
An agarose gel containing the gene of interest
Buffer BL
Buffer PC
Buffer PW
ddH2O
Spin Columns CB2
Collection Tubes 2ml
2ml Centrifuge tube
Pure Ethanol
Procedure
1. Put the agarose gel containing the target gene into the 2ml centrifuge tube, and
add Buffer PC with the same volume as the agarose gel into the centrifuge tube. The tube is incubated in a
water bath at 50°C for 10 minutes and gently turned up and down periodically until the gel is completely
dissolved.
2.Put the spin column CB2 into the collection tube and add 500μl of Buffer BL to the spin column CB2.
Centrifuge the tube at 12,000 rpm for 1 min. Drain the waste liquid in the collection tube, and put the spin
column back into the collection tube.
3. Add the solution obtained in the previous step into the spin column CB2 and centrifuge at 12,000 rpm for
1min. Dump the waste liquid in the collection tube, and put the spin column CB2 into the collection
tube.
4. Add 600μl Buffer PW, to the spin column CB2, centrifuge at 12,000 rpm for 1 min, drain the waste liquid
in the collection tube, and put the spin column CB2 into the collection tube.
5. Repeat Operation 4.
6. Put the spin column CB2 into the collection tube and centrifuge it at 12,000 rpm for 2min. Open the spin
column and place it at room temperature for several minutes, and then dry thoroughly.
7. Put the spin column CB2 into a clean centrifuge tube, and add 30μl ddH2O to the middle position of the
spin film in the air, and leave it at room temperature for 2 min. Centrifuge at 12,000 rpm for 2 min and
collect DNA solution.
Bacterial Cell Plasmid Extraction
Introduction
The program can isolate amplified plasmids. This procedure lyses bacteria at high pH
and uses the spin column to adsorb and purify plasmids in a high concentration salt solution.
Procedure
1. Take 1-5 mL of overnight cultured bacterial solution, add it into the tube.
Centrifuge the tube at 12000 rpm for 1 minute, and pour away the supernatant.
2. Put the spin column into the collection tube, add 500μl Buffer BL to the spin column CP3. Centrifuge the
tube at 12,000 rpm for 1 min, drain the waste liquid in the collection tube, and put the spin column back
into the collection tube.
3. Add 250μl Buffer P1 with RNase A into the centrifuge tube with bacterial precipitation, and completely
suspend bacteria.
4. Add 250μl Buffer P2 into the centrifuge tube and gently turn it up and down 6-8 times to fully lysis the
bacteria.
5. Add 350μl Buffer P3 into the centrifugal tube, immediately turn it gently up and down for 6-8 times to
mix thoroughly, and centrifuge the centrifugal tube at 12,000 rpm for 10 min.
6. Transfer the supernatant collected in the previous step to the spin column CP3 without sucking out the
precipitation. Centrifuge the centrifugal tube at 12,000 rpm for 30-60 s, dump the waste liquid in the
collection tube, and put the spin column CP3 into the collection tube.
7. Add 600μl Buffer PW with pure ethanol into the spin column CP3 and centrifuge the tube at 12,000 rpm for
30-60s. Dump the waste liquid in the collection tube and put the spin column CP3 into the collection
tube.
8. Repeat Step 7.
9. Centrifuge the tube at 12,000 rpm for 2min. Remove the cap of the spin column CP3 and place it at room
temperature for several minutes to thoroughly dry the remaining Buffer PW in the spin column CP3.
10. Place the spin column CP3 in a clean centrifuge tube, and add 50-100μl ddH2O to the middle part of the
spin column CP3. The solution containing the target gene was collected into the centrifuge tube after being
placed at room temperature for 2 min and centrifuged at 12,000 rpm for 2 min.
Assembly of Recombinant Plasmids
Introduction
This program uses recombinase to directionally clone the target gene fragments with
the same sequence at both ends into the linearized plasmid DNA to generate the recombinant plasmid carrying
the target gene.
Procedure
1.Add Ynμl of the NTH insert (n≤5) and Xμl of linearized vector to each tube.
2.Add 5μl of 2$\times$CE Mix to each tube.
3.Add ddH2O to each tube to make the total volume of the mixing system reach 10μl.
4.Mix the mixture system and briefly centrifuge.
5.Incubate the tube in a PCR instrument at 50℃, and cool and store the tubes at 4℃ after completion.
Transformation
1. Defrost the stored DH5α on ice.
2. Add 10μl recombinant plasmid to 100μl DH5α, flick the tube wall to mix well, and let it stand for 30min
on ice.
3. After heat shocking the DH5α competent cells 30s in a water bath at 42℃, cool it on ice immediately for
2-3min.
4. Add 900μl LB liquid medium to DH5α bacterial solution, and shake the bacteria solution at 200-250 rpm for
1h at 37℃.
5. Preheat the corresponding resistant LB solid medium in an incubator at 37℃.
6. Centrifuge the tube at 5000 rpm for 5 min and pour off 900μl of supernatant. Resuspend the cells with the
remaining liquid medium and evenly spread on prewarmed LB solid mediums.
7. Incubate the cells in an incubator at 37℃ for 12-16 hours.
Digestion of Plasmid Vector
Introduction
Use two restriction endonucleases to cleave plasmid vector. These enzymes recognize and bind DNA sequences at specific positions of the plasmid. Two restriction endonucleases interact with these DNA sequences to hydrolyze their phosphodiester bonds and linearizing the plasmid.
Procedure
1. The final reaction system in the tube is Xμl plasmid vector, 5μl rCutsmart Buffer, 1μl each of the two restriction enzymes, and ddH2O (43-X)μl.
2. Place the tube in a water bath for 3h at 37℃.
3. Place the tubes in a metal bath for heat inactivation for 25min, after which store these tubes at 4℃.
* X is the volume of solution required to add 1μg of plasmid DNA
Ligase Reaction for joining DNA Segments
Introduction
This procedure enables reattachment of linearized vectors and inserts. This reaction utilizes T4 ligase to link different linear DNA fragments.
1. The final reaction system in the tube is linearized vector Xμl, 2 $\times$ Universal Ligation Mix 5μl, the NTH insert Ynμl, ddH2O (5-X-Y1-Y2... –Yn)μl.
2. Mix the mixture system and briefly centrifuge.
3. Incubate the tube in a PCR amplifier at 37℃, and cool and store the tubes at 4℃ after completion.
Transformation:This procedure is the same as “Assembly of Recombinant Plasmids” one.
Colony PCR
Introduction
This technique is to test whether bacteria carry recombinant plasmids by PCR reaction. PCR enables primers to bind specifically to plasmids and amplify the gene of interest in several thermal cycles.
Procedure
1. Add 10μL 2$\times$ Taq Master Mix to each tube
2. Add 2μl of forward primer and reverse primer to each tube
3. Add 1μL template DNA to each tube
4. Add 7μL ddH2O to each tube
5. Put the tubes into the PCR Amplifier for PCR
Thermocycling conditions for Colony PCR
| Step |
Temperature |
Duration |
Cycles |
| Initial denaturation |
95℃ |
3min |
1 |
| Denaturation |
95℃ |
15s |
35 |
| Annealing |
(Tm-5)℃ |
15s |
| Extension |
72℃ |
60s/kb |
| Final extension |
72℃ |
5min |
1 |
| Hold |
4℃ |
∞ |
1 |
Electroporation of Pseudomonas aeruginosa
Introduction
This procedure uses electric shock to make competent cells take up recombinant plasmids.
Procedure
1. Defrost the stored competent cells on ice.
2. Add the solution containing 50ng recombinant plasmid to 100μl bacterial solution in the tube, flick and mix well, and then bath on ice for 10 minutes.
3. Inject the bacterial solution into a 2mm electroporation cuvettes and placed in an electroporator for electro-transformation.
4. Transfer the bacterial solution in the electroporation cuvettes to a tube, add 900μl LB liquid medium, and shake the bacteria for 1h at 37℃.
5. Preheat the corresponding resistant LB solid medium in an incubator at 37℃.
6. Centrifuge the tube at 10,000 rpm for 5 min and pour off 900μl of supernatant. Resuspend the cells with the remaining liquid medium and evenly spread on prewarmed LB solid mediums.
7. Incubate the cells in an incubator at 37℃ for 15 hours.
3 Expression
3.1 SDS-Page
3.1.1 Preparation
Bacterial Culture: Shake the bacteria one day in advance, approximately 5 ml.
Lysis Buffer:
1. 50 mM Tris (1.2114 g)
2. 200 mM NaCl (2.3376 g)
3. Adjust pH to 8.0 (cool in an ice water bath to 4°C)
4. Make up to a final volume of 200 ml.
3.1.2 Procedure
Bacterial Lysis
1. Growth Phase: Use bacteria during logarithmic growth (OD: 0.6–0.8).
2. Prepare a blank control with an empty plasmid (no plasmid introduced) and 1 ml of the gene-introduced bacteria.
3. Centrifuge the samples.
4. Discard 900 μl of supernatant.
5. Resuspend the pellet in: 60 μl lysis buffer and 40 μl 5x SDS loading buffer
6. Incubate in a 100°C metal bath for 10 min (open the lid after 1 min to release pressure).
7. Centrifuge at 14,000 x g for 5 min (at 4°C).
Sample Preparation
1. Take 15 μl of the supernatant for loading onto the gel (precast gel).
2. When adding the comb, wrap it with a paper towel to avoid splashes.
Electrophoresis
1. Run the gel at 80 V for 30 min (until bromophenol blue runs off the stacking gel).
2. Then increase to 120 V for 1 h (stop when bromophenol blue is 2 mm from the bottom of the gel).
3. Follow the manufacturer’s instructions for running the precast gel.
SDS-PAGE
1. Remove the fixing solution and add G-250 stain.
2. Stain for 0.5 h on a shaker.
3. Discard the staining solution and add destaining solution.
4. Place on a destaining shaker, changing the solution until the blue background is removed.
5. Store the gel in deionized water, then scan and save the image.
4 Effect Verification
4.1 Degradation Module
4.1.1 Wet-lab degradation
Prepare the PE particle samples:
1.Weigh 0.1 g PE with sizes of 500μm and 3μm, respectively. Collect each set in a 50 mL centrifuge tube.
2.Open the top cap, put the tube under UV light and immerse the sample with 75 % ethanol for 4 hours.
3.Separate the extra ethanol as much as possible, seal the top with sterility parafilm and dry the samples in tubes.
Prepare the bacteria:
1.Culture the bacteria to OD600 = 0.6 ~ 0.7.
2.Centrifugal, discard the LB medium.
3.Add 0.85 % NaCl solution, gently resuspend the bacteria.
4.Centrifugal again, abandon the supernatant.
5.Add 0.85 % NaCl solution again, gently resuspend the bacteria for further usage.
Co-cultivation:
1.Add 15 mL of inorganic salt culture medium into the tube (with dried PE particles inside).
2.Add 30 mL sterilized ddH2O.
3.For the experiment group, add 1 mL prepared bacteria solution (NaCl washed already) per tube; for the reference group, add 1 mL 0.85 % NaCl solution.
4.Seal the top, co-cultivate in the shaker for 10, 15 and 30 days.
Separating the PE particles:
1.Use filter paper to separate the 500μm PE particles, wash the samples twice with 75 % ethanol.
2.For the 3μm PE particles, separate the supernatant as much as possible, take a portion of the samples in the petri dish, and add 75 % ethanol.
3.Dry the samples in the oven.
Examine the samples:
1.For the 500 μm PE, weigh the samples and record.
2.For both the 500 μm and 3 μm PE, conduct FTIR examination and analyze the data.
3.For both the 500 μm and 3 μm PE, conduct SEM examination.
3.1.2 Co-incubation
Bacterial cultivating protocol：
The engineered and wild-type bacteria were cultured at 37℃ and 220rpm in LB broth to DO600=0.7.
Co-incubation protocol：
0.1 g of 5 mm microplastics and 1 ml of bacterial culture diluted to 1$\times 10^{-4}$ were added to a 1.5 ml centrifuge tube and co-incubated at 220 rpm and 37°C for 30 minutes. The co-incubated microplastics were then transferred to another centrifuge tube containing PBS buffer solution. After vortexing for 15 seconds, the microplastics were placed on a microscope slide and observed under an inverted fluorescence microscope.
4.2 Extracellular Electron Transport
4.2.1 Microbial Fuel Cell (MFC) configuration
We used MFC to exam the bioelectricity output of corresponding strains to further investigate whether e-pili can help the bioelectricity output of P. aeruginosa and the contribution of pilA on EET.
Two 200 ml Perspex chambers were separated by a 5$\times$5-cm2 cross-sectional area CMI-7000 cation exchange membrane. The electrode was made of polished graphite (5$\times$8 cm^2, 4 mm thickness) unless stated otherwise. At the beginning of MFC operation, the devices were autoclaved. The anode and cathode chambers were respectively filled by M9 medium with 4 g/L glucose and 1 M KCl, 500 ml of exponential phase culture (OD600≈0.5) was added into the anode chamber. A 1 kΩ resistor was connected to the electrodes, and the voltages were measured by a digital multimeter. Currents were converted from measured voltage through Ohm's law (voltage = current $\times$ resistance).The greater the detected current, the greater the conductivity between the bacteria.
4.2.2 Measuring content of NADH & NAD+
To verify that the introduction of the nqrF gene can reduce the NADH content in bacteria
1.Set up the control group and the experimental group
The control group was untreated Pseudomonas aeruginosa PAO1 and the experimental group was P.aeruginosa PAO1 of the imported plasmid. The P.aeruginosa PAO1 concentration should be the same or close between the control and experimental groups.
2.Preparation of the samples
For suspended cells, about 106 cells were centrifuged at 600 g for 5 minutes, the culture was added 200 μL ice bath pre-cold NADP+ / NADPH extract with 200μL extract per 1 million cells and was gently blown to facilitate the cell lysis; the lysis process was performed at room temperature or on ice. Subsequently, 4℃ was centrifuged at 12,000 g for 5-10 minutes, and the supernatant was used as the sample to be tested.
3.Preparation of the kit
(1) Preparation of NADH standard: absorb 655 μl NADH and dissolve 5mg NADH provided by this kit.10mM NADH standard after properly packaged -80℃ protected from light.
(2) Setting of NADH standard curve: 10 mM NADH standard was diluted with NAD+ / NADH extract into an appropriate concentration gradient, as the initial test, 0, 0.25, 0.5, 1, 2, 4, 6, 8, 10 μM, add 20μL of standard per well, equivalent to 0, 5, 10, 20, 40, 80, 120, 160, 200 pmol of NADH per well.
(3) Preparation of ethanol dehydrogenase working solution: the ethanol dehydrogenase is diluted 45 times with reaction buffer, and 90μL of ethanol dehydrogenase working solution is used for the detection of each standard or sample. Pay attention to the current allocation and use.
4.Sample determination:
(1)Determination of total amount of NAD+ and NADH in sample: draw 20 μL of NAD+ / NADH extract into 96-well plate to test the sample to reduce experimental error.
(2) Determination of NAD+, NADH content in sample or NAD+ / NADH ratio: absorb 50-100μL of sample to be tested in a centrifuge tube and heat on 60℃ water bath or PCR instrument for 30 minutes to decompose NAD+. If it produces insoluble material after heating, take 10,000 g, centrifuge at room temperature or 4℃ for 5 minutes, and absorb 20μL of the supernatant diluted with NAD+ / NADH extract as the sample to be tested into a 96-well plate to reduce the experimental error.
(3) Refer to the table below to set up the blank control hole, standard hole and sample wells using a 96-well plate. Add the ethanol dehydrogenase working solution and mix well.
|
Blank |
Standard |
Sample |
| Samples to be tested |
|
20μL |
20μL |
| NAD+/NADH extract |
20μL |
|
|
| Ethanol dehydrogenase working fluid |
90μL |
90μL |
90μL |
Table 1. Refer to the table below to set up the blank control hole, standard hole and sample wells using a 96-well plate.
(4)37℃ was incubated for 10 min.
(5)Mix the color solution properly, then add 10μL of color solution to each well, and incubate with 37℃ for 10-20 minutes, when orange-yellow formazan will form. Absorbance at 450nm was measured. If the color is light, the incubation time can be extended to 30-60 min.
(6)Calculation of NAD+ / NADH amount in the sample:
A. Calculate the average absorbance of each point in the standard group, minus the absorbance of the blank control group, which is the absorbance of each standard.
B. A standard curve is drawn with the concentration of NADH as the abscissa and the absorbance as the ordinate. Refer to Figure 1 for the test effect of the NADH standard.
Figure 1 Standard curve of NAD+ / NADH NAD+
C. Calculate the total concentrations of NAD+ and NADH or the concentrations of NADH in the cells, tissues, and other samples, from the standard curve. Without 60℃ heating treatment, the concentration of total NAD+ and NADH (NADtotal); 60℃ heating treatment is the concentration of NADH in the sample.
D. To calculate the amount of NAD+ in the sample and the ratio of NAD+ / NADH according to the following calculation formula. At this point, the total amount of NAD+ and NADH can be expressed by the amount of unit cell number or unit tissue weight.
$$[NAD^+] = [NADtotal] - [NADH]$$
$$[NAD^+]/[NADH] = ([NADtotal] - [NADH])/[NADH]$$
4.3 Carbon Dioxide Fixation Module
4.3.1 Cellulose expression verification
Ultivation Conditions for Cellulose Production in Liquid Medium
Hestrin-Schramm medium (HS), which was used as the basic medium for growth and cellulose production, consists of 20 g glucose, 5 g yeast extract L⁻¹, 5 g polypeptone L⁻¹, 2.7 g Na₂HPO₄ L⁻¹, 1.15 g citric acid L⁻¹, and 5.7 g MgSO₄·7H₂O L⁻¹. The pH of the HS medium was adjusted to 5.7 using 1.0 M HCl. E. coli BL21 strains were used for induced expression. The culture media were supplemented with kanamycin (50 mg/mL) to prevent the loss of the pBBR1MCS2 plasmid.
Direct detection of BC in E.coli cultures
Since BC is an extracellular material secreted into the culture medium, it was directly detected 18 hours after cultivation in the cellulose production medium.
We selected BL21 strains containing the plasmid and those without it, preparing three replicates of each in 50 mL conical flasks. These cultures were then incubated statically at 37°C for 18 hours.
Characterizing cellulose production by Congo red visually evaluate the qualitative and quantitative aspects of BC production, Congo red was employed.
Congo red staining on plates for detecting cellulose production
From the plasmid-containing and plasmid-free strains, 2 μL of each was diluted into 100 μL of LB and spread on LB plates containing 0.004% Congo red to determine whether cellulose was produced in the E. coli strains. The plates were incubated at 37°C for 24 hours. Red colonies indicated the binding of Congo red to cellulose. A color change would occur in cellulose-producing colonies due to the growth of cellulose fibers on the LB agar plates.
Congo red staining for detecting cellulose production by measuring liquid absorbance
To reduce error, each sample was subjected to Congo red staining three times, and the corresponding cellulose production for the change in absorbance was calibrated using the recovered bacterial cellulose.
1.Take 2 mL of a 14-15 hour cell culture (grown in HS medium) with similar cell concentrations for all samples.
2.Centrifuge at 5000 rpm for 10 minutes.
3.Resuspend the cells in 1 mL of a 1% tryptone solution containing 40 mg of Congo red and incubate on a shaker for 2 hours at 220 rpm and 37°C.
4.Remove the bound Congo red by centrifugation at 17,000$\times$g for 5 minutes, and calculate the amount of unbound Congo red by measuring the absorbance of the supernatant at 490 nm.
Recovery and weighting of bacterial cellulose
To minimize error, each group was performed three times, using bacteria in the same growth state each time.
1.After culturing for 24 hours in HS medium, centrifuge at 12,000 rpm for 5 minutes.
2.Discard the supernatant and resuspend in a 1% NaOH solution (w/v), then treat at 70 °C for 20 minutes.
3.Centrifuge at 12,000 rpm for 1 minute.
4.Discard the supernatant, resuspend in deionized water, and centrifuge again at 12,000 rpm for 1 minute. Repeat this step three times.
5.Dry the samples in microcentrifuge tubes at 50 °C until constant weight is achieved.
6.Weigh and record the data.
Fourier transform infrared (FTIR) analysis
The recovered cellulose was used for Fourier transform infrared (FTIR) analysis.
The spectral characteristics of the BC produced by GM strains exhibit very strong absorption bands between 900 and 1243 cm⁻¹, attributed to the C–O and C–O–C stretching vibrations of glucose. The band corresponding to the stretching vibrations of –OH groups in cellulose is located between 3500 and 3100 cm⁻¹. The spectral band between 2800 and 2900 cm⁻¹ is assigned to the C-H stretching vibrations of BC (including -CH₂ and -CH₃), while the spectral bands located between 1420 and 1278 cm⁻¹ correspond to the in-plane bending of C-H groups. Cellulose synthesis is correlated with biofilm formation in E. coli, which is an extracellular polymeric substance composed of extracellular DNA, proteins, and polysaccharides. Another notable region of the spectra appears around 1517 and 1537 cm⁻¹, corresponding to amide I groups in this substance; furthermore, the band at 1640 cm⁻¹ is assigned to –OH bending.
4.3.2 Measuring content of NADPH & NADP+
To verify that import of nadM and nadK genes can increase the intracellular NADPH content
(1) Set up the control group and the experimental group
The control group was untreated E.coli BL21 and the experimental group was E.coli BL21 of the imported plasmid. The E.coli BL21 concentration should be the same or close between the control and experimental groups.
(2) Preparation of the samples
For suspended cells, about 106 cells were centrifuged at 600 g for 5 minutes, the culture was added 200 μL ice bath pre-cold NADP+ / NADPH extract with 200μL extract per 1 million cells and was gently blown to facilitate the cell lysis; the lysis process was performed at room temperature or on ice. Subsequently, 4℃ was centrifuged at 12,000 g for 5-10 minutes, and the supernatant was used as the sample to be tested.
(3) Preparation of the kit
A. Preparation of NADPH standard: 1mM NADPH standard is obtained after absorbing 6ml of ultrapure water for 5mg NADPH provided by this kit.
B. Setting of NADPH standard curve: dilute 1 mM NADPH standard with NADP+ / NADPH extract into an appropriate concentration gradient, set concentrations of 0, 0.25, 0.5, 4.5, 1, 2, 4 μM for the initial test, add 50μL of standard per well, equivalent to 0, 1, 2.5, 25, 20, 10, 10, 20, 200 ml of NADPH per pmol per well.
C. Preparation of G6PDH working solution: dilute G6PDH 50 times with reaction buffer. Testing of each standard or sample requires 100μL of G6PDH working solution.
(4) Sample determination
A. Determination of total amount of NADP+ and NADPH in samples: draw 50μL of the tested sample diluted with NADP+ / NADPH extract into a 96-well plate and set repeat wells for the sample to reduce experimental error.
B. Determination of NADP+, NADPH content or NADP+ / NADPH ratio: absorb 100-200 μL of sample to be tested in a centrifuge tube and heat on 60℃ bath or PCR for 30 minutes to decompose NADP+.
C. Refer to the table below to set up the blank control hole, standard hole and sample wells using a 96-well plate. Add G6PDH working solution and mix well.
|
Blank |
Standard |
Sample |
| Samples to be tested |
|
20μL |
20μL |
| NADP+/NADPH extract |
20μL |
|
|
| G6DPH working fluid |
90μL |
90μL |
90μL |
Table 2. Refer to the table below to set up the blank control hole, standard hole and sample wells using a 96-well plate.
D.37℃ was incubated for 10 min.
E. Mix the color solution properly, then add 10 μL of the color solution to each well, and incubate for 37℃ for 10-20 minutes, at which point orange formazan will form. Absorbance at 450 nm was measured. If the color development is light, the incubation time can also be appropriately extended to 30-60 minutes. As the incubation time increases, the color development will gradually deepen.
(5) Calculation of the NADP+ / NADPH amount in the sample
A. Calculate the average absorbance of each point in the standard group, minus the absorbance of the blank control group, which is the absorbance of each standard.
B. A standard curve is drawn using the concentration of NADPH as the abscissa and the absorbance as the ordinate. Refer to Figure 2 for the detection effect of the NADPH standards. If the incubation time is too long, the color development of the high concentration standard will reach the platform. At this time, the standard product should be selected to draw the standard curve, or the absorbance data of the standard product with a short incubation time should be selected to draw the standard curve.
Figure 2 Standard curve of NADP+ / NADPH NADP+
C. Total NADP+ and NADPH concentrations or NADPH concentrations in cells, tissues, and other samples were calculated from the standard curve. Without 60℃ heating treatment, the calculated concentration of the total amount of NADP+ and NADPH in the sample (NADPtotal) is the concentration of NADPH in the sample.
$$[NADP^+] = [NADPtotal] - [NADPH]$$
$$[NADP^+]/[NADPH] = ([NADP_{total}] - [NADPH])/[NADPH]$$
4.4 Safety Module
4.4.1 The plasmid anti-loss function
(1)Make three tubes of 1 mL of bacterial solution of E. coli DH5α with empty plasmid pMV using non-resistant LB broth as control group, and make three tubes of 1 mL bacterial solution of E. coli DH5α with plasmid pMV-hok/sok using the same medium as the experimental group. Both groups are placed on the shaking bed at 37℃ to culture and conduct cell passaging every 10h. At least ten passages can be transferred to the solid media.
(2)After reaching the standard of transference, dilute the bacterial solution of the both groups to $10^{-6}$ times of the primary concentration, and 50 μL of each tube is taken to transfer to the solid medium of non-antibiotic LB. All the plates are placed in the incubator, invertedly cultivating for 12h at 37℃.
(3)Pick up the single colonies with good growth on each plate after the culture completes and delineate them on the plates with ampicillin in control groups and experimental groups respectively. Culture the plates in the incubator at 37℃ for 6h to observe the growth status of each single colony.
4.4.2 The function of PopdH
(1)Prepare Pseudomonas aeruginosa PAO1 with vector pAB-PopdH-GFP in LB broth containing ampicillin, expanding at 37°C until the OD600 of the bacterial solution is between 0.6 and 0.7.
(2)Prepare 1M disinfected citrate solution. Take 7 portions of 1 mL of the bacterial solution above, and separately add citrate solution to form a citrate concentration gradient of 0mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM. At the same time, the LB broth with ampicillin is configured as a series of citrate concentration gradient of 0mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM as a background for reserve.
(3)All 7 groups are incubated in a shaker at 37°C for 2h. Take some of bacterial solution and its corresponding citrate concentration of broth to conduct the measurement of green fluorescence intensity.
4.4.3 The effect of the suicide system
(1)Prepare 0.1M IPTG solution and add it to the LB broth with ampicillin, configuring it as LB-Amp medium with 0.6mM IPTG for reverse.
(2)Take 5 mL of the broth above. Divide Pseudomonas aeruginosa PAO1 with plasmid pAB-hoksok into 6 groups, which are all placed in the shaker for 12h at 37°C.
(3)Three of the groups are set as control groups, and 100 μL of each bacterial solution is reintroduced into 5 mL of LB-Amp medium with 0.6 mM IPTG; the other three groups are set as experimental groups, and 100 μL of each bacterial solution is reintroduced into 5 mL of LB-Amp medium without IPTG. All six groups are incubated in a shaker at 37°C for 18h.
(4)Examine OD600 of the bacterial solution of each group after cultivation.
