Team:CSU CHINA/Experiments

PROTOCOLS AND EXPERIMENTS

Experiments

Synechocystis PCC6803 Protocols

1. prepare a stock solution

Chemical Amount (g)
\(100\ \times\ BG11 (per L)\)
\({NaNO}_{3}\) 149.6
\({MgSO}_{4}\) ·7 \({H}_{2}O\) 7.49
\({CaCl}_{2}\) ·2 \({H}_{2}O\) 3.6
Citric acid 0.6
Add \(1.12\ ml\ 0.25\ M\ {Na}_{2}EDTA\), \(pH\ 8.0\ 0.25\ M\)
\({Na}_{2}EDTA\), \(pH8.0\ (per\ 100 ml)\)
\({Na}_{2}EDTA\) 9.3
Trace elements \((per\ 100 ml)\)
\({H}_{3}{BO}_{3}\) 0.286
\({MnCl}_{2} ·4\) \({H}_{2}O\) 0.181
\({ZnSO}_{4}\ ·7\) \({H}_{2}O\) 0.022
\({Na}_{2}{MoO}_{4}\ ·2\) \({H}_{2}O\) 0.039
\({CuSO}_{4}\ ·5{H}_{2}O\) 0.008
\({Co(NO3)}_{2}\ ·6\ {H}_{2}O\) 0.005
Iron stock \((per\ 100ml)\)
Ferric ammonium citrate 1.11
Phosphate stock \((per\ 100ml)\)
\({K}_{2}{HPO}_{4}\) 3.05
\({Na}_{2}{CO}_3\) stock \((per\ 100ml)\)
\({Na}_{2}{CO}_3\) 2
TES buffer, \(pH 8.2 (per\ 100ml)\)
TES 22.9
\({NaHCO}_{3}\) stock \((per\ 100 ml)\)
\({NaHCO}_{3}\) 8.4
HEPES, \(pH 8.2 (per\ 500 ml)\)
HEPES 119.15
Vitamin B12 \((Per 50 ml)\)
Cyanocobalamin 0.02

2. Prepare BG11 medium

  • 2.1. Prepare stock solutions of \(100\ \times\ BG11\), trace elements and iron stock in protocol1.
  • 2.2. Prepare separate solutions of phosphate stock, \({Na}_{2}{CO}_3\) stock, N-[Tris (hydroxymethyl) methyl]-2-aminoethanesulfonic acid (TES) buffer and \({NaHCO}_{3}\) in protocol1.
  • 2.3. Autoclave the phosphate and \({Na}_{2}{CO}_3\) stocks—Filter-sterilize TES buffer and \({NaHCO}_{3}\) with \(0.2 µm\) filters.
  • 2.4. Prepare BG11 by combining 976 ml of water, \(10 ml\) of \(100\ \times\ BG11\), \(1 ml\) of trace elements and \(1 ml\) of iron stock and autoclave the solution. After this solution has cooled to room temperature, add 1 ml of phosphate stock, 1 ml of \({Na}_{2}{CO}_3\) stock and 10 ml of \({NaHCO}_{3}\) .
  • 2.5. For BG11 solid medium, add 15 g of agar and \(700 ml\) of water to one flask. To the second flask, add \(3 g\) of \({Na}_{2}{S}_{2}{O}_{3}\), \(226 ml\) of water, \(10 ml\) of \(100\ \times\ BG11\), 1 ml of trace elements and 1 ml of iron stock. Autoclave both solutions. After these solutions have cooled to room temperature, combine them and add 1 ml of phosphate stock, 1 ml of \({Na}_{2}{CO}_3\) stock, 10 ml of TES buffer, and 10 ml of \({NaHCO}_{3}\) . Note: Solutions are prepared separately to avoid precipitation of certain salts.

3. Culture Synechocystis PCC6803 and refresh medium

  • 3.1. Culture: the Synechocystis was placed under the condition of \(30℃\), \(40 E/m2/s\) light, with fresh BG11 culture medium \({OD}_{730}\)>0.3, to avoid the occurrence of photoinhibition), refresh medium every two days.
  • 3.2. refresh medium: 30-50mL of Synechocystis was taken and centrifuged at 2300g for 5 min. The cell precipitation was washed twice with a fresh BG11 medium. The final suspension was either in the original volume of fresh BG11 culture medium (for preserving the original growth state) or in a larger volume of BG11 culture medium (for propagation).

4. Determine the growth of Synechocystis PCC6803

  • 4.1. 1mL Synechocystis was taken from the culture medium and placed into a cuvette.
  • 4.2. Turn on the spectrophotometer (UNICOSH, UV2355) and preheat it for 20 mins.
  • 4.3. Choose the wavelength as 730nm.
  • 4.4. Cuvette filled with distilled water must be scanned as a blank to provide a spectral background.
  • 4.5. The growth condition was determined by \({OD}_{730}\)

5. Cyanobacteria (Synechocystis) conjugation and transfer

  • 5.1. 2 mL culture medium of cyanobacteria in the logarithmic phase \({OD}_{730}\) is close to 0.7) was taken and centrifuged at 6000 rpm for 5 min. The cell precipitation was washed twice with a fresh BG11 medium. Finally, resuspend it in 200 L of BG11 medium.
  • 5.2. E. coli strain E. coli HB101 (transferred with pRL443+pRL623+ plasmid) cultured overnight was transferred to a fresh LB medium for the logarithmic period (\({OD}_{600}\) approximately 1.0).
  • 5.3. 2 mL E. coli in the logarithmic phase was taken and centrifuged at 6000 rpm for 5 min. The cell precipitation was washed twice with fresh LB medium. Finally, they were resuspended in 200 L of LB medium.
  • 5.4. Cyanobacteria suspension and Escherichia coli cell suspension were mixed. The Cyanobacteria -E. coli suspension was placed at 30℃, 30~50 E/m2/s and stood for 4 h.
  • 5.5. The Cyanobacteria -E. coli suspension (100~200 L per plate) was coated on the BG11 plate covered with cellulose nitrate film (NC film). 20 hr was required under 30℃, 30~50 E/m2/s light conditions.
  • 5.6. Then, the NC membrane was transferred to a solid BG11 plate containing antibiotics (concentration) and continued to be cultured under 30℃, 30~50 E/m2/s light conditions.
  • 5.7. After a few days (4-7 days), there are single colonies of Cyanobacteria growing up; The single colonies were selected from solid plates containing antibiotics and then separated for cell passage at 30℃, 30~50 E/m2/s.

Experiment protocols

1. prepare a stock solution

Chemical Amount (g)
\(1g/L\) \({Cd}_{2+}\) stock \((per\ 50mL)\)
\(3{CdSO}_{3}•8\ {H}_{2}O\) 0.1141
6% sodium alginate \((per\ 100mL)\)
sodium alginate 6
4% calcium chloride \((per\ 100mL)\)
calcium chloride 4

2. Cadmium ion determination (Shanghai Youlong Biotech Co .Ltd, BE0241)

  • 2.1. Take \(100μL\) of medium supernatant and add 300μL of sample extract, mix well. That is the sample solution to be tested.
  • 2.2. Add \(50µL\) of cadmium ion standards (0, 0.4, 1.2, 3.6, 10.8, 32.4ppb) to each standard well, and add 50µL of sample solution to the sample well.
  • 2.3. Add \(50μL\) of antibody solution and \(50ul\) of enzyme label solution to both standard wells and sample wells, shake gently to mix, and then incubate at 25°C for 30min.
  • 2.4. After the incubation, shake off the liquid in the microwells, add about \(250μL\) of diluted washing solution to each well, leave it for 1 minute, and discard the liquid. Repeat the washing 4 times. Pour out the liquid and pat dry the plate on absorbent paper or dry towel.
  • 2.5. Add \(100 µL\) of the color developer to each well, shake gently to mix, and develop color in a constant temperature incubator at 25°C for 15 minutes.
  • 2.6. After the color reaction is over, add \(100μL\) stop solution to each well. Measure at 450nm with a microplate reader. It must be completed within 10 minutes after adding the stop solution.

3. Cadmium tolerance and absorption capacity test of wild type Synechocystis PCC6803 under different cadmium concentrations

The Synechocystis (\({OD}_{730}\)=0.7) were treated with 0.1,0.5,1,2,5,10 (mg/L) cadmium concentration gradients.

  • 3.1. Centrifuge and the collected Synechocystis was resuspended in 30mL fresh BG11 culture medium until \({OD}_{730}\)=0.5. Then the Synechocystis grew to 0D730=0.7 after 36h culture.
  • 3.2. After absorbance was determined, 3, 15, 30, 60, 150, and 300μL of 1g/L \({Cd}_{2+}\) stocks were added to corresponded groups.
  • 3.3. Samples were taken at 0.5h, 1h, 1.5h, 2h, 4h, 12h, 24h and 48h to determine the concentration of cadmium ions in the culture medium (Shanghai Youlong Biotech Co .Ltd, BE0241) and the growth of Synechocystis (\({OD}_{730}\)).

4. Determination of cadmium absorption ability of different densities of Synechocystis PCC6803 under high and low cadmium conditions

Five groups of Synechocystis (\({OD}_{730}\)=0.3,0.6,0.8,1.1,1.4) were treated with 0.1mg/L (low cadmium group) and 2mg/L (high cadmium group), respectively.

  • 4.1. 30mL of fresh BG11 culture medium was used to resuspense Synechocystis to \({OD}_{730}\)=0.2, 0.4, 0.6, 0.8, 1.2. Then the Synechocystis grew to target \({OD}_{730}\) after 36h culture.
  • 4.2. After \({OD}_{730}\) was determined, 3 μL and 60 μL of 1mg/L \({Cd}_{2+}\) mother liquor were added to the corresponded group.
  • 4.3. Samples were taken at 0.5h, 1h, 1.5h, 2h, 4h, 12h, 24h and 48h to determine the concentration of cadmium ions in the culture medium (Shanghai, youlong biological company, BE0241) and the growth of algae (\({OD}_{730}\)).

5. Embedding and fixation of Synechocystis PCC 6803

  • 5.1. The logarithmic growth stage was taken, centrifuged at 2300g for 5min, and washed twice with fresh BG11 medium. Finally, it was resuspended in a fresh BG11 culture medium until \({OD}_{730}\)=0.7.
  • 5.2. The above Synechocystis was mixed with 6% sodium alginate 1:1. The mixed liquid was then dropped into 4% calcium chloride with a dripping device to produce 3.5mm calcium alginate microbean. Curing at 4℃ for 3h.
  • 5.3. The 10 microbeans were dissolved in 10 mL sodium citrate solution with a mass fraction of 2%. The absorbance of the dissolved solution at 730 nm was determined as the biomass density of Synechocystis. Ten microbeans of calcium alginate were dissolved in the sodium citrate solution as the colorimetric control solution.

6. Influence of calcium alginate microbeans on cadmium absorption

The cadmium removal ability of the three calcium alginate microbean groups containing Synechocystis was compared by 0.1mg/L (low cadmium group) and 2mg/L (high cadmium group).

  • 6.1. Experimental group 1: The Synechocystis (\({OD}_{730}\)=0.7) mixed with 6% sodium alginate 1:1 was blown well. The mixture was dropped into 4% calcium chloride with a self-made device to produce 3.5mm calcium alginate microbeans. Curing at 4℃ for 3h. Put 50 microbeans in a 30mL volume medium.
  • 6.2. Experimental group 2: BG11 medium and 6% sodium alginate 1:1 were mixed and blown well. The mixed liquid was dropped into 4% calcium chloride with a device to produce 5mm glueballs. Curing at 4℃ for 3h. Put 50 microbeans in a 30mL volume medium.
  • 6.3. Experimental group 3: Put 5mL Synechocystis (\({OD}_{730}\)=0.7)(equal to 50 microbeans) in 30mL volume.
  • 6.4. Samples were taken at 0.5h, 1h, 1.5h, 2h, 4h, 12h, 24h and 48h to determine the concentration of cadmium ions in the culture medium (Shanghai youlong biological company, BE0241) and the growth of Synechocystis (\({OD}_{730}\)).

7. express parts in E.Coli and their reaction to cadmium

  • 7.1. The E. coli cells, harboring pVZ322-parts, were grown overnight in LB medium containing corresponded antibiotics. Different flasks containing 20 ml LB were inoculated with this overnight culture. The cells were incubated at 37℃ in a shaker till \({OD}_{600}\) of 0.7.
  • 7.2. After an hour, 0.1mg/L of cadmium was added to cultures. Control cultures containing E. coli cells transformed with only pVZ322 vector (without insert) were also treated in the same way as to test cultures.
  • 7.3. The \({OD}_{600}\) was measured in 400μL bacterial medium taken every 30min, by which the growth curve was drawn.
  • 7.4. Another 400 L bacterial medium was taken every 30min too and centrifuged at 8000rpm for 2min to determine cadmium concentration.

General Molecular Protocols

1.PCR Reaction

Reagent Volume(μl)
5X Buffer 10
DMSO 4
dNTP 4
Forward Primer 2
Reverse Primer 2
Targeted DNA fragment 1
Phusion 1
dd\({H}_{2}O\) 26
In Total 50

Program:

\(98℃(5min)\)

\(\left. \begin{array}{l} \text{98℃}&\left(30s\right)\\ \text{58℃}&\left(30s\right)\\ \text{72℃}&\left(30s/kb\right) \end{array} \right\} *40\)

\(72℃\ (10min)\)

\(4℃\)

2. Gel recycling（Omega）

  • 2.1. Agarose EB gel was prepared, and electrophoresis was used to separate DNA fragments.
  • 2.2. After enough time has passed, the required DNA fragments are carefully cut off under ultraviolet light. And try to get rid of the excess gel.
  • 2.3. The weight of the empty centrifuge tube was weighed, and the gel with the target segment was cut off and placed in the 1.5mL centrifuge tube and weighed. The weight of the gel block was calculated, and its volume was approximately determined. A binding Buffer of the same gel volume was added, and the mixture was placed in a water bath of 55℃~65℃ for 7min to melt completely, during which the gel was mixed every 2-3 minutes.
  • 2.4. Transfer 700 μl of DNA-agarose solution to a HiBindTM DNA column and place the column in a clean 2ml collection tube. Centrifuge at 10,000×g for 1min at room temperature.
  • 2.5. Put the column back into the collection tube and add 300 μL Binding Buffer; Centrifuge at 10,000×g for 1 min at room temperature, then discard the filtered liquid.
  • 2.6. Put the column back into the collection tube and add 700 μL SPW Wash buffer. Centrifuge at room temperature 10,000×g for 1 minute, then discard the filtered solution.
  • 2.7. Put the column back into the collection tube and repeatedly add 700μ L SPW Wash buffer. Centrifuge it at 10,000×g for 1 minute at room temperature to discard the filtered solution.
  • 2.8. Discard the liquid and reinsert the empty column back into the collection tube. Centrifuge it at 10,000×g for 1min to dump the remaining liquid.
  • 2.9. The column was placed on a clean 1.5 mL centrifuge tube and added with a 50 μL sterilized water column film. The column was centrifuged at 10,000×g for 1 minute.

3. Assembly Reaction ( abm, Pro Ligation-Free Cloning Kit)

  • 3.1. Quantify the insert and vector DNA by a UV spectrophotometer or by comparison of the target band against a known molecular weight marker run on the same gel.
  • 3.2. Set up the following reaction on ice:
Reagent Cloning Reaction Example Reaction
2X Pro Ligation-Free MasterMix 10 µl 10 µl
Linearized Vector 3:1 insert-vector molar ratio 60 ng (1 kb insert)
Nuclease Free \({H}_{2}O\) Up to 20 µl Up to 20 µl

To calculate amount of insert to add:

insert (ng)= 3*insert (bp)/ vector (bp)* vector (ng)

  • 3.3. For 1 insert, incubate at 50°C for 15 minutes. For multiple inserts or complicated assemblies, incubate at 50°C for 1 hour. 4
  • 3.4. Transform immediately or store samples at -20°C until transformation.

4. transfection of competent cells (DH5a CELL and HB101 CELL)

  • 4.1.Put the cells on ice and let stand for 20min, then precool the EP tube, transfer 20μL of cells to the EP tube;
  • 4.2.The 2 μL of plasmid was added to the cells and placed on the ice for 30min;
  • 4.3.Heat shock 42℃, the 90s, followed by 5min on ice;
  • 4.4.Add 500 mL of antibiotic-free liquid medium and incubate in a shaking table at 37℃ for 60min
  • 4.5.coat plates, select monoclonal colony or add to 6mL LB medium added with 6μL antibiotics (\(1000×\))

5. protein extraction

  • 5.1. Solutions and reagents: lysis buffers
    • NP-40 buffer
      • 150 mM NaCl
      • 1.0% NP-40 (possible to substitute with 0.1% Triton X-100)
      • 50 mM Tris-HCl, pH 8.0
      • Protease inhibitors
    • RIPA buffer (radioimmunoprecipitation assay buffer)
      • 150 mM NaCl
      • 1% IGEPAL CA-630
      • 0.5% sodium deoxycholate
      • 0.1% SDS (sodium dodecyl sulphate)
      • 50 mM Tris-HCl, pH 8.0
      • Protease inhibitors
    • Tris-HCl
      • 20 mM Tris-HCl
      • Protease inhibitors
  • 5.2. Centrifuge centrifugal tube containing E.Coli or Synechocystis in a centrifuge at 4°C.
  • 5.3. Washed with PBS and then resuspend cells in lysis buffer (equal to the volume of precipitate) in a microcentrifuge tube.
  • 5.4. Maintain constant agitation for 30 min at 4°C.
  • 5.5. Centrifuge in a microcentrifuge at 4°C. 12000rpm, 10min.
  • 5.6. Gently remove the tubes from the centrifuge, place it on the ice, aspirate the supernatant, put it in a fresh tube kept on ice, and discard the pellet.

6. Western blot

  • 6.1. Remove a small volume of lysate to perform a protein quantification assay. Determine the protein concentration for each cell lysate.
  • 6.2. Determine how much protein to load and add a 5×SDS sample buffer.
  • 6.3. To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 10 min.
  • 6.4. Run the gel for 1–2 h at 100 V.
  • 6.5. The membrane can be either nitrocellulose or PVDF. Activate PVDF with methanol for 1 min and rinse with transfer buffer before preparing the stack. The time and voltage of transfer may require some optimization. We recommend following the manufacturer’s instructions. Transfer of proteins to the membrane can be checked using Ponceau S staining before the blocking step.
  • 6.6. Block the membrane for 1 h at room temperature or overnight at 4°C using a blocking buffer.
  • 6.7. Incubate the membrane with appropriate dilutions of primary antibody in blocking buffer.
  • 6.8. Wash the membrane in three washes of TBST, 5 min each.
  • 6.9. Incubate the membrane with the recommended dilution of conjugated secondary antibody in blocking buffer at room temperature for 1 h.
  • 6.10. Wash the membrane in three washes of TBST, 5 min each.
  • 6.11. For signal development, follow the kit manufacturer’s recommendations. Remove excess reagent and cover the membrane in transparent plastic wrap. Acquire images using darkroom development techniques for chemiluminescence or standard image scanning methods for colorimetric detection.