Culture Medium Preparation

LB liquid medium

Reagent: LB broth powder( Sangon Biotech A507002-0250 ), deionized water

Device: Measuring cylinder, Electronic balance, Medicine spoon

Procedure(for 1 L volume):

  1. Accurately weigh 25 g of LB broth powder and then add 1 L of deionized water.
  2. High-pressure steam sterilization at 121℃ for 20 min.

Note: If antibiotics need to be added, they must be added after autoclaving, otherwise the antibiotics will be ineffective. Antibiotics need to be filtered separately for sterilization .

 

Component

Tryptone

10g/L

Yeast extract

5g/L

NaCl

10g/L

 

LB plate

Reagent: LB broth powder(Sangon Biotech A507002-0250), deionized water , A gar powder (HuanKai Microbial 028990A) .

Device: Measuring cylinder, Electronic balance, Medicine spoon

 

Procedure(for 1 L volume with 1.5% agar):

1. Accurately weigh 25 g of LB broth powder , 15g agar powder and then add 1 L of deionized water.

2.High-pressure steam sterilization at 121℃ for 20 min.

Note: If a plate with antibiotics is required, the antibiotics should be added after autoclaving and before the liquid begins to set, and thoroughly mixed . Antibiotics need to be filtered separately for sterilization .

Component

Tryptone

10 g/L

Yeast extract

5 g/L

NaCl

10 g/L

A gar powder

15 g/L

 

BG11 liquid medium

Reagent: BG11 (Coolaber AML201-100L) , deionized water

Device: Measuring cylinder, Electronic balance, Medicine spoon

Procedure(for 1L volume ):

  1. A dd 988ml of deionized water and then add 10ml ingredient A , 1ml ingredient B, 1ml ingredient C of BG11 (Coolaber AML201-100L).
  2. High-pressure steam sterilization at 121℃ for 20 min.

Note: If antibiotics need to be added, they must be added after autoclaving, otherwise the antibiotics will be ineffective. Antibiotics need to be filtered separately for sterilization .

Do not mix ingredient A, ingredient B, ingredient C directly, or it will precipitate

Component

NaNO 3

1500 mg/L

K 2 HPO 4

40 mg/L

MgSO 4

36.6 mg/L

CaCl 2

27.2 mg/L

Citric acid

6 mg/L

Ammonium ferric citrate

6 mg/L

EDTANa 2

1 mg/L

Na 2 CO 3

20 mg/L

H 3 BO 3

2.86 mg/L

MnCl 2 ·4H 2 O

1.81 mg/L

ZnSO 4

0.22 mg/L

CuSO 4 ·5H 2 O

0.08 mg/L

Na 2 MoO 4 ·2H 2 O

0.39 mg/L

CoCl 2 ·6H 2 O

0.0409 mg/L

 

 

BG11 plate

Reagent: BG11 (Coolaber AML201-100L) , deionized water , A gar powder(HuanKai Microbial 028990A).

Device: Measuring cylinder, Electronic balance, Medicine spoon

Procedure(for 1L volume with 1.5% agar ):

  1. A dd 988ml of deionized water and then add 10ml ingredient A , 1ml ingredient B, 1ml ingredient C of BG11 (Coolaber AML201-100L) and then add 15g agar powder.
  2. High-pressure steam sterilization at 121℃ for 20 min.

Note: If a plate with antibiotics is required, the antibiotics should be added after autoclaving and before the liquid begins to set, and thoroughly mixed . Antibiotics need to be filtered separately for sterilization .

Do not mix ingredient A, ingredient B, ingredient C directly, or it will precipitate .

Component

NaNO 3

1500 mg/L

K 2 HPO 4

40 mg/L

MgSO 4

36.6 mg/L

CaCl 2

27.2 mg/L

Citric acid

6 mg/L

Ammonium ferric citrate

6 mg/L

EDTANa 2

1 mg/L

Na 2 CO 3

20 mg/L

H 3 BO 3

2.86 mg/L

MnCl 2 ·4H 2 O

1.81 mg/L

ZnSO 4

0.22 mg/L

CuSO 4 ·5H 2 O

0.08 mg/L

Na 2 MoO 4 ·2H 2 O

0.39 mg/L

CoCl 2 ·6H 2 O

0.0409 mg/L

A gar powder

15 g/L

 

 

CoB BG11 liquid medium

Reagent: BG11 (Coolaber AML201-100L) , deionized water , NaCl , NH 4 Cl, sucrose.

Device: Measuring cylinder, Electronic balance, Medicine spoon

Procedure(for 1L volume ):

  1. A dd 988ml of deionized water and then add 10ml ingredient A , 1ml ingredient B, 1ml ingredient C of BG11 (Coolaber AML201-100L).
  2. Add 6.2g NaCl , 0.214g NH 4 Cl , and 20g sucrose .
  3. High-pressure steam sterilization at 121℃ for 20 min.

Note: If antibiotics need to be added, they must be added after autoclaving, otherwise the antibiotics will be ineffective. Antibiotics need to be filtered separately for sterilization .

Do not mix ingredient A, ingredient B, ingredient C directly, or it will precipitate .

Component

NaCl

6.200g/L

NH 4 Cl

0.214g/L

S ucrose

20g/L

NaNO 3

1500 mg/L

K 2 HPO 4

40 mg/L

MgSO 4

36.6 mg/L

CaCl 2

27.2 mg/L

Citric acid

6 mg/L

Ammonium ferric citrate

6 mg/L

EDTANa 2

1 mg/L

Na 2 CO 3

20 mg/L

H 3 BO 3

2.86 mg/L

MnCl 2 ·4H 2 O

1.81 mg/L

ZnSO 4

0.22 mg/L

CuSO 4 ·5H 2 O

0.08 mg/L

Na 2 MoO 4 ·2H 2 O

0.39 mg/L

CoCl 2 ·6H 2 O

0.0409 mg/L

 

 

 

Transformation and Coating

  1. C entrifuge the synthesized target plasmid dry powder sample (2μg) in a tube at 12000rpm for one minute.
  2. Add 200μL ddH 2 O, close the tube cover, and vortex shock to dissolve it fully.
  3. Take 100 μL (1 tube) receptive cells melted on ice, add 5μL dissolved target plasmid (50ng), gently mix, and leave on ice for 30 min.
  4. 42 ℃ water bath heat shock 45~60 s, quickly transferred to the ice bath, standing for 2 min (do not shake the sample during the ice standing process, otherwise it will reduce the conversion efficiency).
  5. A dd 700 μL LB medium without antibiotics into the centrifuge tube, mix it well, place it at 37 ℃ and 200 rpm for 60 min.
  6. Absorb 100μL resuscitation solution and evenly apply it to LB medium containing kanamycin, and place the plate upside down in 37 °C incubator for overnight culture.
  7. S elect several single colonies for confirmation by PCR amplification.

7.1 Culture the selected single colonies overnight using LB medium containing kanamycin.

7.2 Take 1ml of the bacterial solution cultured overnight, centrifuge it at 8000rpm for 3 minutes, remove the supernatant, suspend it with 30 μ l NaOH (20 mM), then add 70 μ l Tris (1M), mix it well, and take it as the template DNA.

7.3 Set up the PCR system on the ice.

Green Taq Mix(Vazyme P131)

12.5 μ L

Forward Primer (10uM)

1 μ L

Reverse Primer (10uM)

1 μ L

Sterile ddH 2 O

9.5 μ L

Template DNA

1 μ L

Total

25 μ L

7.4 Set up the PCR program, put the PCR tube into the instrument, and start to run.

 

95 ℃ ， 3min

95 ℃ ， 15s

30 ×       55 ℃ ， 15s

72 ℃ ， 60s

72 ℃ ， 5min

4 ℃ ， forever

Note: The annealing temperature should be adjusted according to the Tm of the primers, and the extension time should be adjusted according to the length judged by the amplification.

  1. Verification by agarose gel electrophoresis.

8.1 Weigh 0.36g agarose, add 30ml 1 × TAE solution (1.2%), heat to dissolve agarose.

8.2 Add 3uL SYBR dye(biosharp BS354B), mix it well, pour into the gel tank and cool at room temperature.

8.3 Add 2μl DNA marker (biosharp BL102A) into the loading pore. Add 5 μl PCR product into the loading pore. Record the position of the sample added.

8.4 Turn on the electrophoresis apparatus and start running.

8.5 After running, check the result on the gel imager, take a photo and record.

8.6 Sent the PCR products to a genetic company for sequencing and verification.

 

 

Plasmid assembly

  1. C entrifuge the synthesized target plasmid dry powder sample (2μg) in a tube at 12000rpm for one minute.
  2. Add 200μL ddH 2 O, close the tube cover, and vortex shock to dissolve it fully.
  3. Working on ice, add the following reagents.

10X cutsmart

5 μ L

Plasmid （ 2 μ g ）

40 μ L

EcoRI-HF

2.5 μ L

XbaI

2.5 μ L

Total

50 μ L

Note: The enzyme activity of EcoRI-HF and XbaI is 20000 unit/ml. 1U is the restriction enzyme amount required for complete enzyme digestion of 1μg DNA within 1h at 37℃ in a 50ul system. Using 2.5 μL of the restriction enzyme is equivalent to 50 units, or 50μg of DNA per hour.

  1. Mix it well and place it in an incubator at 37℃ for more than 3h.
  2. C onfigured the agarose gel, then electrophoresis, and f ind the target fragment after digestion based on the band position.

Note: The target fragment length of sorAB is 1942bp and the target fragment length of SQR-SDO is 7139bp. The difference between the target fragment of SQR-SDO and the plasmid fragment is only 22bp, and the target fragment can be isolated by reducing the agarose ratio, extending the electrophoresis time, and using the plasmid fragment comparison.

  1. Precisely cut the gel containing the target bands with a scalpel under the UV transilluminator.
  2. Accurately weigh the pieces of gel. Put them into 1.5 ml EP tubes respectively.
  3. According to the instructions in the Gel Extraction Kit(OMEGA D2500-01) recover the DNA fragment until the last step.
  4. Take the PCR tube and add the reagent according to the following ratio.

Pet 28-SQR-SDO （ 7139bp ）

(0. 03 - 0. 08 pmol)

sorAB （ 1942bp ）

(0.1 - 0.3 pmol)

Sterile purified water

Up to 5 μ L

Note: The molar ratio of the carrier to the insertion sequence needs to be between 1:2 and 10, and the specific volume needs to be adjusted according to the recovered DNA concentration.

  1. Add 1 volume (10 μL) of Solution I (DNA Ligation Kit <Mighty Mix >(Cat. #6023)) and mix completely.
  2. Incubate at 16℃ overnight .
  3. After incubation, transform the assembled plasmid into E . coli DH10B, marked the plate with antibiotics, and screened out the successfully introduced plaque.
  4. S elect the single clone for PCR amplification and confirmation, and sent the PCR product to a gene company for sequencing verification .
  5. After verification, the amplified Plasmid was extracted using the NucleoSpin® Plasmid and Mini kit .
  6. The extracted plasmid DNA was stored in a -20 ° C refrigerator .

 

Formaldehyde indication system experiment

  1. Selecte verified E. coli strains and inoculated them into LB medium containing kanamycin, culture them at 37 ℃ and 200 rpm.

2. When the growth of E. coli reaches the early exponential stage (OD about 0.4), add 100ul into the 96-well black transparent bottom plate, and then add methanol to the final concentration of 0, 5, 10, 25, 50, 100, 250, 500, 1000 μmol/L, respectively. Make three parallels for each concentration gradient.

3. Add 100ul wild-type E. coli of the same early exponential stage (OD about 0.4) as the background fluorescence blank and 100ul sterilized LB liquid medium as the OD600 blank. Make three parallels for each concentration gradient.

4. Teste the fluorescence intensity and OD600 every 10min with multifunctional microplate reader. Set the temperature 37 ℃ and the maximum speed in multifunctional microplate reader.

 

F ormaldehyde metabolism system Experiments

  1. Take 9 1.5ml EP tubes and configured formaldehyde gradient solution with final concentrations of 0, 5, 10, 20, 50, 100, 200, 500, 1000µM, 350ml each. Note: LB medium was used to configure formaldehyde gradient solution.

2. Set up the experimental group: E. coli BL21 with Metabolically associated plasmids; Control group: wild type E. coli BL21; Blank group: LB. E. coli was cultured in conical bottles to the stable stage, and add formaldehyde to the final concentration of 500 µmol/L and cultured at 37℃ and 200rpm.

3. Take 600µl bacterial solution every 5 or 10min. Immediately return the cone to the shaker after sampling.

4. Centrifuge them at 13000 rpm for 2 min, and transfer 350µl supernatant to 1.5ml EP tube as test liquid.

5 . Add 350µl acetyl acetone color developing solution(OKA GCTC6108) to each sample, mix well, heat in a constant temperature water bath at 40℃ for 30min, remove and cool at room temperature for 10min.

6. Measure OD412 on microplate reader , making three parallels for each sample.

7. Use the data of hole sample s which have determine the formaldehyde concentration to prepare a standard curve, and calculated the formaldehyde concentration of other hole samples according to the standard curve.

8. According to the formaldehyde content of different time, make the formaldehyde content change diagram of different samples.

 

hydrogen sulfide metabolism system Experiments

  1. Take 9 1.5ml EP tubes and configured Na 2 S gradient solution with final concentrations of 0, 0.5, 1, 2.5, 5, 10, 25, 50, 100 µg/ml, 550ml each. Note: LB medium was used to configure formaldehyde gradient solution.
  2. Set up the experimental group: E. coli BL21 with Metabolically associated plasmids; Control group: wild type E. coli BL21; Blank group: LB. E. coli was cultured in conical bottles to the stable stage, and add Na 2 S to the final concentration of 50 µ g/ml and cultured at 37℃ and 200rpm.
  3. Take 1000µl bacterial solution every 30 min. Immediately return the cone to the shaker after sampling.
  4. Centrifuge them at 13000 rpm for 2 min, and transfer 150µl supernatant to 1.5ml EP tube as test liquid.
  5. Hydrogen sulfide detection kit (bioleaper BR5000292) reagents are added to each sample in the following order .
    1. Add 150 µ L of reagent 1 and 150 µ L of reagent 3 to each sample and mix thoroughly.
    2. Add 150 µ L of reagent 4 and mix thoroughly , let stand at 4 ℃ for 10 minutes.
    3. Add 30 µ L of reagent 5, thoroughly shake and mix, and let stand at room temperature for 20 minutes .
  6. Measure OD 665 on microplate reader, making three parallels for each sample.
  7. Use the data of hole sample s which have determine the Na 2 S concentration to prepare a standard curve, and calculated the Na 2 S concentration of other hole samples according to the standard curve.
  8. According to the Na 2 S content of different time, make the formaldehyde content change diagram of different samples.

Sucrose decomposition system Experiments

  1. Take 8 1.5ml EP tubes and configured sucrose gradient solution with final concentrations of 0, 1.0, 2.5 , 5 , 10 , 15, 20, 25 mg/ml , 60ml each. Note: LB medium was used to configure formaldehyde gradient solution.
  2. Set up the experimental group: E. coli BL21 with plasmid pET28a-SacC ; Control group: wild type E. coli BL21; Blank group: LB. E. coli was cultured in conical bottles to the stable stage, and add sucrose to the final concentration of 20mg/mL, add IPTG to the final concentration of 1 mM and then cultured at 37℃ and 200rpm.
  3. Take 100µl bacterial solution every 1.5h. Immediately return the cone to the shaker after sampling.
  4. Centrifuge them at 13000 rpm for 2 min, and transfer 60µl supernatant to 1.5ml EP tube as test liquid.
  5. Plant Sucrose Content Assy kit (boxbio AKPL006M) reagents are added to each sample in the following order .
    1. Add 30µl of reagent 1, mix thoroughly , incubate at 95℃ for 5 minutes, and cool to room temperature.
    2. Add 420ul reagent 2 and 120µl reagent 3 , mix thoroughly, incubate at 95℃ for 30 minutes, and cool to room temperature.
  6. Measure OD 480 on microplate reader, making three parallels for each sample.
  7. Use the data of hole sample s which have determine the sucrose concentration to prepare a standard curve, and calculated the sucrose concentration of other hole samples according to the standard curve.
  8. According to the sucrose content of different time, make the formaldehyde content change diagram of different samples.