Notebook

Experimental Diary

2023.6.16 Yidong Wang , Jiulin Chen

Preparation of LB solid medium 400ml *1 ： 10g LB + 6g Agar + 400ml ddH 2 O

Preparation of LB liquid medium 400ml *1 ： 10g LB + 400ml ddH 2 O

 

Formaldehyde solution preparation

Formaldehyde mother solution (37 wt.%) MW ： 30.03 ； concentration ： 1 * 37% / 30.03 / 1 = 12.321mol/L

Formaldehyde stock solution ( 1mol/L ) ： 8.13ml m other solution + 91.87ml ddH2O

 

Preparation of kanamycin mother solution

Weigh 500mg kanamycin solid in a 15ml centrifuge tube and add deionized water to 10ml ( 50mg/mL )

U se a filter membrane with a pore size of 0.22 μ m to filter the mother solution , and divided it into 10 1.5ml EP tubes, which were stored in the refrigerator at -20 ℃ .

 

Preparation of LB solid medium containing kanamycin

Heat the prepared 400ml LB solid medium until the agar is completely melted and cool at room temperature . When cool to not hot (about 50°C), add 400μl kanamycin mother liquor (50μg/mL), mix well, and pour the medium into a disposable plate for use.

 

Plasmid introduction of pET28a-FrmR-fdh-fghA &pET28a-FrmR-sfGFP

1. C entrifuge t he synthesized target plasmid dry powder sample (2μg) at 12000rpm for 1 minute.

2. A dd 200μ l ddH2O ， c lose the tube cover and vortex to dissolve it fully 。

3. Take 1 tube (100 μl ) of competent cell s and place them on ice for 20-30 min to melt them , a dd 5μl dissolved target plasmid (50ng), mix gently, 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. Add 700 μL of LB medium without antibiotics into the centrifuge tube, mix, incubate at 37 ℃ at 200 rpm for 60 min.

6. A bsorbed 100μL culture solution, evenly coat it on LB medium containing kanamycin, and place the plate upside down in an incubator at 37 °C for overnight culture .

Note: Plate 1, 1 (H) is the imported pET28a-FrmR-sfGFP engineering bacteria, and 2, 2 (H) is the imported pET28a-FrmR-fdh-fghA engineering bacteria

 

2023.6.17 Yidong Wang , Jiulin Chen

Prepare 100 μmol /L formaldehyde solution in a watering can: 1μl stock solution + 10 mL deionized water

 

Plate fluorescence test

Plate 1 (H) was sprayed with 100 μ M formaldehyde three times and incubated at 37 ℃ for 1h. Results: No obvious fluorescence was observed .

Plate 1 (H) added 100 μ M formaldehyde 100 μ L and incubated at 37 ℃ for 3h.

Results: No obvious fluorescence was observed .

Plate 1 (H) added 100 μ M formaldehyde 100 μ L and incubated at 37 ℃ for 4.5 h.

Results: No obvious fluorescence was observed .

Plate 1 (H) was simultaneously added with 1mM, 10mM, 100mM formaldehyde 10 μ l and incubated at 37 ℃ overnight.

 

PCR verification

S elect 2 single colonies from each plate and name them 1a, 1b, 1c, 1d, 2a, 2b, 2c, 2d. Dissolve in 20μ L sterile water as PCR template.

P repare the PCR system on ice.

Taq PCR Master Mix	12.5 μ L
Forward Primer (10uM)	1 μ L
Reverse Primer (10uM)	1 μ L
Sterile ddH 2 O	9.5 μ L
T emplate	1 μ L
Total	25 μ L

Note: Primers for 1, 1 (H) were pET28-R &28aF; Primers for 2 , 2 (H) were pET28-R &pET28-F

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

 

95 ℃ ， 3min

95 ℃ ， 15s

30 × 55 ℃ ， 15s

72 ℃ ， 60s / 100s

72 ℃ ， 5min

4 ℃ ， forever

Note: 1, 1 (H) extension time is 60s; 2, 2 (H) extension time is 100s .

 

Verification by agarose gel electrophoresis

Weigh 0.72 g agarose, add 60ml 1×TAE solution (1.2%), heat to dissolve agarose. Add 6μL SYBR dye, pour into the mold, cool at room temperature. Absorb 5µL PCR product, add it to the gel hole, and turn on the electrophoresis apparatus. After the agarose gel electrophoresis, view the results on the photometer and photograph .

R esult : There are multiple strips and they do not match the length of the target fragment.

 

Prepar e formaldehyde in various concentrations : 100mM , 10mM , 1mM , 100μM , 10μM

 

Plate streak

S treak s ingle colonies 1a, 2b, 1c with inoculating loop on LB plates containing kanamycin

 

2023.6.18 Yidong Wang

Streak single colonies 1a, 2b, 1c again ， a nd perform liquid culture with 10mL tube (4mL LB + 4 μL kanamycin mother solution ).

 

P reparation of culture medium

LB （ s olid ） 400ml *1

LB （ l iquid ） 200ml *2

 

R ename the single colony 1a, 2b, 1c plates marked by plates the previous day as 1aI, 2bI, 1cI, and perform liquid culture with 10ml tube (4ml LB + 4μL kanamycin mother solution ).

 

2023.6.19 Yidong Wang , Zhiang Chen

PCR verification

Samples ： 1a ， 2b ， 1c ， 2d ， 1aI ， 2bI ， 1cI

T a k e 100μL of liquid culture bacteria solution, centrifuge at 12000rmp for 1min, remove the supernatant, add 30μL NaOH solution and resuspend, and then add 70μL Tris as template.

P repare the PCR system on ice.

Taq PCR Master Mix	12.5 μ L
Forward Primer (10uM)	1 μ L
Reverse Primer (10uM)	1 μ L
Sterile ddH 2 O	9.5 μ L
T emplate	1 μ L
Total	25 μ L

Note: The primers used were pET28-R &28aF

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

 

95 ℃ ， 3min

95 ℃ ， 15s

30 × 55 ℃ ， 15s

72 ℃ ， 60s / 100s

72 ℃ ， 5min

4 ℃ ， forever

Note: 1, 1 (H) extension time is 60s; 2, 2 (H) extension time is 100s .

 

Verification by agarose gel electrophoresis

Result: The test was successful. Seven samples showed a single band, which was consistent with the target size.

P reservation ： A bsorb 700μL of liquid culture solution + 300μL of 50% glycerol for culture preservation and store it in a -80 ℃ refrigerator .

 

Pre-experiment of green fluorescence induced by formaldehyde

Take 28 1.5ml EP tubes and add them to 1ml LB medium and 1μL kanamycin mother solution, then inoculate 7 tubes with 1μL 1a, 1c, 1aI, 1cI bacterial solution at 37 ° C and 200rpm. W hen OD600 ≈ 0. 4, Add formaldehyde to concentrations as shown in the following table ：

S ample	1a	1c	1aI	1cI
Formaldehyde concentration （ μ M ）	0	0	0	0
	5	5	5	5
	10	10	10	10
	25	25	25	25
	50	50	50	50
	100	100	100	100
	250	250	250	250

Continue cultivation at 37 degrees, 200rpm. Every 10min, observe the green fluorescence by naked eye under ultraviolet lamp.

Results: The difference of 4 samples began to appear after 30min, and the difference was obvious after 1h, and the fluorescence intensity increased with the concentration gradient of formaldehyde. The difference between samples 1aI and 1c is the most obvious.

 

2023.6.20 Yidong Wang , Zhiang Chen

M ade the bacterial solution of sample 1aI induced by 250μM formaldehyde yesterday into a temporary plate and observed under a fluorescence microscope , we found that there was obvious fluorescence .

 

2023.6.21 YidongWang

S treak strain 1aI a nd 1c.

 

2023.6.29 Yidong Wang , Jiulin Chen , Xin Yang

Culture s train 1aI in a conical flask, 100ml LB medium +100μL kanamycin mother solution, and inoculated with 100μL 1aI activation solution . Culture at 37 ° C, 200rpm for 2-3h, take 100ul to black transparent bottom 96-well plate, add 1mmol/L formaldehyde 0.5μl, 1μl, 2.5μl; 10mmol/L formaldehyde 0.5μL, 1μL, 2.5μl; 100 mmol/L formaldehyde 0.5μL, 1μL . The final concentrations were 0, 5, 10, 25, 50, 100, 250, 500, 1000 μmol /L. Make three parallels for each concentration gradient. Use 100μ L of the same length to the early index stage of Escherichia coli bacteria containing no fluorescent gene as background fluorescence blank and use 100μ L of sterilized LB liquid medium as OD600 blank. Make three parallels . M easure t he fluorescence intensity and OD600 every 10min with an ELIASA . Set the temperature 37 ℃ and the maximum speed in the ELIASA .

Results: The lid of the b lack transparent bottom 96 - well plate was fog ged , detection failure.

 

2023.6.30 Jiulin Chen , Xin Yang

  Culture s train 1aI in a conical flask, 100ml LB medium +100μL kanamycin mother solution, and inoculated with 100μL 1aI activation solution.

  Carry out t he fluorescence indication experiment of formaldehyde .

Results: The temperature was set incorrectly and the experiment failed .

 

2023.7.03 Yidong Wang

A ctivate strain 1aI in 4m L LB liquid medium .

 

2023.7.04 YidongWang

Activate strain 1aI in 4m L LB liquid medium .

Activate strain BL21 ( No plasmid was introduced ) i n 4mL LB liquid medium a s a negative control .

 

2023.7.05 Jiulin Chen , Xin Yang

Culture strain 1aI , BL21 in a conical flask, 100ml LB medium +100μL kanamycin mother solution, and inoculated with 100μL 1aI , BL21 activation solution.

Carry out t he fluorescence indication experiment of formaldehyde .

Result: The experiment succeeded .

 

2023.7.06 YidongWang Zhiang Chen Shuang Liang

T est the formaldehyde metabolism of strains 2b, BL21 and LB medium .

Results: Strain 2b proved to be able to metabolize formaldehyde, but the metabolic capacity was smaller than that of BL21 .

 

2023.7.13 Yidong Wang

I noculate 2b ， BL21 .

 

2023.7.15 Yidong Wang , Zhiang Chen , Shuang Liang

I ncrease the initial formaldehyde concentration to 500μM, and repeated the formaldehyde metabolism test for strains 2b, BL21 and LB .

Results: The metabolic capacity of strain 2b was still lower than that of BL21.

 

2023.7.23 Yidong Wang , Xin Yang

R eceive Polycoccus PCC7942 strain from Shanghai University of Science and Technology .

Spectral scanning of the bacterial solution revealed an absorbance peak at 685nm.

 

Prepare BG11 medium 800ml *1

 

I noculate 100μL PCC7942 bacterial solution in 4ml BG11 medium in a light incubator at 30 ℃ and 1500lux.

P our the plate BG11 BG11+Kan

 

2023.7.24 Yidong Wang

PCC7942 coating , LB plate, 37 ℃ ， cultured in the dark

BG11 30 ℃ ， cultured in the light

 

2023.7.25 Yidong Wang , Zhiang Chen , Shuang Liang

Repeat t he formaldehyde metabolism test for strains 2b, BL21 and LB .

Results: The metabolic capacity of strain 2b was still lower than that of BL21 .

 

2023.7.26 Yidong Wang

P repare medium: LB (liquid) 400ml*1

Prepare sodium alginate solution ： 100ml d dH 2 O + 2.0g s odium alginate

 

2023.7.27 Yidong Wang

P repare medium: CoB BG11 liquid medium

S treak + Liquid culture : 1aI, 1c

 

2023.7.28 Yidong Wang , Jiulin Chen

D etecte t he absorbance of PCC7942 OD685 and 1aI, 1c OD600 .

 

Sodium alginate embedding first attempt

10ml sodium alginate solution + 1ml 1aI b acterial solution

S lowly drop t he mixtur e into 0.2M sterilized CaCl2 solution using a syringe with the needle removed , and le ave for 5h to make the sodium alginate embedment fully calcify . Then equally divided the embedding into LB medium and CoB BG11 medium at 25 ℃ and 200rpm for culture.

The bacteria solution of PCC7942 coated on July 24 did not grow on BG11 medium, but plaque grew on LB medium cultured in the dark, indicating that the cyanobacteria bacteria solution was contaminated by stray bacteria. S elect single colony for streaking.

Prepare 0.2M PBS 50ml* 2, r emove bacteria by filtration

D issolve sodium alginate embeddings with PBS according to protocol method and measured OD600.

 

2023.7.29 Jiulin Chen

M easure the OD600 of 1aI in LB medium and CoB BG11 medium respectively.

 

2023.7.30 Jiulin Chen

Measure the OD600 of 1aI in LB medium and CoB BG11 medium respectively.

 

2023.7.31 Jiulin Chen

Measure the OD600 of 1aI in LB medium and CoB BG11 medium respectively.

 

2023.8.01 Jiulin Chen

Measure the OD600 of 1aI in LB medium and CoB BG11 medium respectively.

 

2023.8.02 Jiulin Chen

Measure the OD600 of 1aI in LB medium and CoB BG11 medium respectively. A lso measured t he pH value of the medium .

 

2023.8.03 Jiulin Chen

Measure the OD600 of 1aI in LB medium and CoB BG11 medium respectively. A lso measured t he pH value of the medium .

C onduct plasmid transformation experiments on PCC7942 according to protocol.

 

2023.8.04 Jiulin Chen

Measure the OD600 of 1aI in LB medium and CoB BG11 medium respectively. A lso measured t he pH value of the medium .

Prepare BG11 plate 200ml*1

Heat, add 200u L kanamycin, pour plate

 

2023.8.05 Jiulin Chen

Take 100u L of the transformed bacterial solution and coated it on BG11 plate and cultured it in a light incubator.

 

2023.8.18 Yidong Wang , Jiulin Chen

Prepare medium

LB plate 400ml*1

LB plate 200ml*1

LB liquid medium 200ml*1

BG11 plate 500ml*1

BG11 liquid medium 200ml*1

I noculate the transformed strain ABC on 8. 3 into 4ml BG11+4μL Kan and culture it with light .

I noculate 1ml of the strain inoculated on 7.23 into 4ml BG11 and cultured it by light .

P our plate

 

2023.8.19 Yidong Wang

C onduct secondary purification of PCC7942 contaminated strains growing on LB medium .

I ntroduce t he SacC plasmid and CobFDB plasmid into BL21 according to protocol.

 

2023.8.20 Yidong Wang

Import failed the day before. No colony has grown on the plate.

 

2023.8.21 Yidong Wang

Sodium alginate embedding second attempt

10ml Sodium alginate solution +1ml 1aI bacterial solution

D rip the calcium chloride solution and incubate it in 4ml LB medium and 4ml CoB BG11 medium after standing for 5h.

 

2023.8.22 Yidong Wang

A dd formaldehyde into yesterday's sodium alginate embedding culture system to 250 and 500μM and continued to culture at 37 ℃ and 200rpm for 1h. Place the vibrating tube under ultraviolet light for observation , i t was found that E. coli coated with sodium alginate could be successfully induced by formaldehyde to emit green fluorescence.

Import SacC and CobFDB back to BL21.

 

2023.8.23 Yidong Wang , Jiulin Chen

C olon ies grew on the plate. S elect ed two single colonies from the Escherichia coli plate with SacC plasmid , streaked and named as Sa and Sb . Select t wo single colon ie s on the E. coli plate with CobFDB plasmid , streaked and named Ca and Cb .

Try again to introduce the CscB plasmid into PCC7942.

Prepare 0.1M sodium bicarbonate solution and filter to remove bacteri a.

Prepare 0.1M c alcium chloride solution and filter to remove bacteri a.

Inoculate 1ml of PCC7942 into 4ml BG11 + 0.8ml 0.1M sodium bicarbonate solution .

 

2023.8.24 Yidong Wang

Sa ， Sb ， Ca ， Cb w ere successfully verified by PCR .

Streak Sa ， Sb ， Ca ， Cb

C oat PCC7942 on BG11 &BG11+Kan

 

Inoculate PCC7942 with 50μL bacterial solution in 4ml BG11 + 1ml 0.1M sodium bicarbonate solution + 5μL kanamycin mother solution ; a nd 50μL bacterial solution in 4ml BG11 + 1ml sodium bicarbonate solution of 0.1M.

I noculate Sa in 4ml LB+4μL kanamycin mother solution.

Inoculate S b in 4ml LB+4μL kanamycin mother solution.

 

2023.8.25 Yidong Wang

Inoculate Sa in 4ml LB+4μL kanamycin mother solution.

Inoculate Sb in 4ml LB+4μL kanamycin mother solution.

Inoculate BL21(DE3) i n 4ml LB .

 

2023.8.26 YidongWang , Xin Yang

Preserve Sa ， Sb ， Ca ， Cb

C onduct sucrose decomposition efficiency experiment ( S ample ： BL21 、 Sa 、 Sb ) . See protocol for details .

 

Inoculate Sa in 4ml LB+4μL kanamycin mother solution.

Inoculate Sb in 4ml LB+4μL kanamycin mother solution.

Inoculate BL21(DE3) i n 4ml LB.

Inoculate PCC7942-1 i n 4ml LB.

Inoculate PCC7942- 2 i n 4ml LB.

 

2023.8.27 Yidong Wang

Inoculate Sa *2 in 4ml LB+4μL kanamycin mother solution.

Inoculate BL21(DE3) i n 4ml LB.

 

2023.8.28 Yidong Wang , Xin Yang

Conduct second sucrose decomposition efficiency experiment ( S ample ： BL21 、 Sa 、 Sa + IPTG ) . See protocol for details .

 

2023.9.04 Yidong Wang

Sor , SQR-SDO Plasmid enzyme digestion, ligand assembly . See protocol for details.

Select the target strip, conduct gel extraction . See protocol for details.

 

2023.9.05 Yidong Wang

C onduct gel extraction again . See protocol for details.

T est t he formaldehyde metabolism of strains Ca, Cb , BL21 and LB .

Results: The metabolic capacity of strain Ca and Cb was still lower than that of BL21 .

 

2023.9.06 Yidong Wang

Inoculate Strain Cb in LB medium with formaldehyde concentration of 500M to screen out strains with strong formaldehyde resistance (high expression of formaldehyde metabolism genes).

 

Inoculate AB ( sorAB ) puncture bacteria in 4ml LB+4μL kanamycin mother solution.

Inoculate SS ( SQR-SDO ) puncture bacteria in 4ml LB+4μL kanamycin mother solution .

 

DNA is connected overnight for 16 hours

 

2023.9.07 Yidong Wang

I ntroduce t he connected plasmid into Escherichia coli DH10B, electro revolution *2, calcium revolution *1 . See protocol for details.

I ntroduce p lasmid m (H2S indicator plasmid) into Escherichia coli BL21, calcium revolution.

 

Extract the DNA of PCC7942-1 &2 DNA . Perform PCR with bacterial universal primers 27F and 1942R, and send the PCR product to sequencing companies for sequencing.

Results: The contaminated strain was nitrate-reducing bacterium .

 

Inoculate Cb 4ml *2 .

2023.9.08 Yidong Wang

9.7 Conversion result :

E lectro revolution : Only a few colonies have grown

C alcium revolution : Colony count is normal

Plasmid m: Colony count is normal

 

S elect electro revolution colony, name it SA1 ( electro ) . S treak + 4ml liquid culture

S elect calcium revolution colony, name it SA 1 ( Ca 2+ ) &SA2 ( Ca 2+ ) . S treak + 4ml liquid culture

Select m colony, name it M1 & M2 . Streak + 4ml liquid culture

 

Inoculate s train Cb to a new LB medium with formaldehyde concentration of 500M to screen out the strains with strong formaldehyde resistance (high expression of formaldehyde metabolism genes).

 

2023.9.09 Xin Yang

P reserve SOCE316 * 3 PCC7942 contaminated strain —— nitrate - reducing bacteria

 

2023.9.12 Yidong Wang

PCR verification . S ample : SA1 ( Ca 2+ ), SA2 ( Ca 2+ ) , M1 , M2 . Succeeded.

 

Inoculate PCC7942 i n BG11+NaHCO 3 medium , and measure OD685.

BG11+NaHCO 3 m edium ( BG11 ： 0.1M NaHCO 3 = 4:1 )

 

  Inoculate PCC7942 in BG11+NaHCO 3 m edium and add with different concentrations of ampicillin to detect the growth. ( Amp : 25, 50, 75, 100 mg/L )

 

2023.9.14 Yidong Wang

Measure PCC7942 bacterial solution OD685

Inoculate M1 , M2 , SA1 ( Ca 2+ ), SA2 ( Ca 2+ )

Culture s train Cb in a new LB medium with formaldehyde concentration of 1000M to screen out the strains with strong formaldehyde resistance (high expression of formaldehyde metabolism genes).

 

2023.9.15 Yidong Wang

Measure PCC7942 bacterial solution OD685

PCC7942 second conversion experiment . See protocol for details.

M 1 &M2 gradient S 2- concentration indication test

Divid M1 and M2 bacterial solution into 1ml in 1.5ml EP tube, and add Na 2 S solution until the final concentration was 10 , 1, 0.1, 0 μ g/ml. Mix well and culture overnight at 37 ℃ .

 

2023.9.16 Yidong Wang

Measure PCC7942 bacterial solution OD685

Prepare LB liquid medium 100ml *10

Prepare BG11 plate 200ml *2

 

Simplify the H 2 S detection process

Simplified process ：

①        150 μ l liquid to be tested + 150 μ l reagent 1 + 150 μ l reagent 1 , thoroughly mix

②        Add 1 50 μ l reagent 4, thoroughly mix

③        Add 30 μ l reagent 5 , Leave for 20 minutes , m easure OD665

Results: After simplification: 1.799Abs Original process: 1.800Abs

 

9.15 M1 &M2 gradient S 2- concentration indication test

Prepare t he bacteria solution into temporary plates and observed under fluorescence microscope. No red fluorescence was found.

 

2023.9.17 Yidong Wang

Measure PCC7942 bacterial solution OD685

 

Extract plasmid . S ample ： SA1 , SA2 . See protocol for details.

Result ：

	ng/ μ l	A260/280	A260/230
SA1	73.9	2.04	2.32
SA2	85.9	2.07	2.32

 

2023.9.19 Yidong Wang , Zhiang Chen

Formaldehyde metabolism test . S ample ： Cb , Cb+Kan , LB , BL21 . Succeed.

 

Measure PCC7942 bacterial solution OD685

 

2023.9.20 Yidong Wang

Re-inoculate PCC7942 BG11 + Na2CO3 to a concentration of 1.2g/L

Measure PCC7942 bacterial solution OD685

 

2023.9.22 Yidong Wang

Measure PCC7942 bacterial solution OD685

The cyanobacteria added with Na 2 CO 3 in 9.20 did not grow . I noculated with BG11+ NaHCO3 to a concentration of (0.02M).

Inoculate M1 &M2

 

2023.9.23 Yidong Wang

M1 &M2 gradient S 2- concentration indication second test

Divid M1 and M2 bacterial solution into 1ml in 1.5ml EP tube, and add Na 2 S solution until the final concentration was 10 , 1, 0.1, 0 μ g/ml. Mix well and culture overnight at 37 ℃ .

 

2023.9.25 Yidong Wang

Measure PCC7942 bacterial solution OD685

Inoculate Sa & Sb

 

2023.9.26 Yidong Wang

Measure PCC7942 bacterial solution OD685

P repare CoB + m other solution ：

Weigh 6.200g NaCl ， 0.214g NH 4 Cl ， 2g Sucrose ， Dissolve in 50ml sterile water and filter to remove bacteri a . Add 50ml/L when using.

 

2023.9.27 Yidong Wang

PCC7942 third conversion experiment . See protocol for details.

Measure PCC7942 bacterial solution OD685

 

2023.9.28 Yidong Wang

Measure PCC7942 bacterial solution OD685

Name t he transformed PCC7942, PA, PB, PC, coat them on BG11 plate with kanamycin added, and inoculate in liquid culture with kanamycin added, at 30 ℃ , with supplementary light culture.

 

2023.9.30 Yidong Wang

Measure PCC7942 bacterial solution OD685

Inoculate SS 、 M1 、 M2

 

2023.10.02 Yidong Wang

Measure PCC7942 bacterial solution OD685

SS &M1 &M2 coculture S2 indication test

Add 450μl SS bacterial solution and 450μl M1 bacterial solution into 1.5ml EP tube, a dd IPTG until the concentration is 1mM , add Na 2 S until the concentration is 10, 1, 0.1, 0 μ g/ml ， o vernight culture at 37 ℃ .

Add 450μl SS bacterial solution and 450μl M 2 bacterial solution into 1.5ml EP tube, a dd IPTG until the concentration is 1mM , add Na 2 S until the concentration is 10, 1, 0.1, 0 μ g/ml ， o vernight culture at 37 ℃ .

 

2023.10.03 Yidong Wang

Measure PCC7942 bacterial solution OD685

Prepare t he SS-M1\SS-M2 bacterial solution into temporary plates and observed under fluorescence microscope.

Results: A small amount of fluorescence was observed in SS-M2 (10μg/ml), and the rest was not observed.

Place t he co-culture solution in a shaker for further culture.

 

2023.10.04 Yidong Wang

Measure PCC7942 bacterial solution OD685

Prepare t he SS-M1\SS-M2 bacterial solution cultured for 2 days into temporary plates and observed under fluorescence microscope.

Results: A small amount of fluorescence was observed at each concentration gradient.

 

2023.10.05 Yidong Wang

Measure PCC7942 bacterial solution OD685

 

2023.10.06 Yidong Wang

Measure PCC7942 bacterial solution OD685

 

2023.10.07 Yidong Wang

Measure PCC7942 bacterial solution OD685

T he plasmid PCC7942 was transformed successfully, and the colony was successfully grown in the medium containing kanamycin.

Experiment of cyanobacteria-Escherichia coli co-culture system .

Mix cyanobacteria and E. coli in 10ml sodium alginate solution . See protocol for details.

 

2023.10.08 Yidong Wang

Measure PCC7942 bacterial solution OD685

M easure OD of cyanobacteria and Escherichia coli in the co-culture system and sucrose concentration and pH in the medium.

 

2023.10.09 Yidong Wang

Measure PCC7942 bacterial solution OD685

Measure OD of cyanobacteria and Escherichia coli in the co-culture system and sucrose concentration and pH in the medium.