Team:NCHU Taichung/Protocol

Protocol

Making okara solution

1. Mix okara and d.d.water with weight 8：1, and put the mixture into a serum bottle.
2. Prepare a clear serum bottle and sterilize with (1.) together.
3. Use steamer cloth to filter the okara solution.
4. Use tissue and suction filter to filter again.
5. Adjust okara solution to pH 8 with NaOH and HCl.
6. Use .22filter filter again, and get the okara solution.

Making cellulase solution

1. Culture the bacteria Cip A, Xyn C, Cel A, and Cel K in LB broth for 16 hours.
2. Use .22 filter to filter each bacteria, and get the suspension.
3. Store in a 4 ℃ refrigerator.

Making rice straw solution

1. Put straw powder 2 g, NaOAc(pH 5, 3 M) 3 ml, d.d.water 57 ml, Cip A 5.714 ml, Xyn C 22.857 ml, Cel A 5.714 ml, Cel K 5.714 ml, and tetracycline 0.1 ml.
2. Put (1) into a 60 ℃ incubator, and shake for 24 hours.
3. Use tissue and suction filter to filter the solution.
4. Use .22filter filter again, and get the rice straw solution.

Making saccharified straw liquid

1. Put 20% rice straw liquid and 80% okara liquid together.
2. Adjust the solution to pH 8 with NaOH(KOH) and HCl.
3. Filter by .22 filter, and get the saccharified straw liquid.

Enzyme activity (DNS)

1. Mix rice straw solution and DNS reagent for 1：1, and put them into an eppendorf.
2. into a 100℃ dry bath incubator for 10 minutes.
3. Cool to room temperature.
4. Measure 570nm OD value, and convert the glucose, concentration, and enzyme activity.

Transformation of Bacillus subtilis

1. Pre-culture B.subtilis for 15-17 hours.
2. Add 900μl TFI, 25μl 2% CAA and 50μl pre-cultured bacterial solution and incubate at 37°C for 4 hours.
3. Take 900 μl of TFII, add 100 μl of the above TFI bacterial solution, and incubate at 37°C for 1 hour and 15 minutes.
4. Take 100 μl of the above TFII bacterial solution, add 5-6 μl of the plasmids and incubate at 37°C for 1 hour.
5. Add 300μl of LB broth to the mentioned bacterial solution after transformation, and incubate at 37°C for 1 hour.
6. Take 200 μl of the above bacterial liquid and apply it on a solid culture medium and use antibiotics to screen successful strains.

Growth Curve measurement

1. Activate strain within 2ml LB medium containing 1/1000 kanamycin under 37⁰C and constant shaking speed 150rpm overnight.
2. Subculture in flask within 200ml LB medium under 37⁰C and constant shaking speed 150rpm,adjust the initial OD600 value to 0.01.
3. Measure the OD600 value every one hour.

Polymerase chain reaction for PQQ gene

1. Mix the components.
2. Transfer to a PCR machine with the block preheated to 95°C
3. Begin thermocycling.
4. Evaluate amplified DNA by agarose gel electrophoresis followed by ethidium bromide staining.

Polymerase chain reaction for 16s rDNA sequencing

All reaction components on ice

1. Mix the components.
   
2. Transfer to a PCR machine with the block preheated to 95°C
3. Begin thermocycling.
4. Evaluate amplified DNA by agarose gel electrophoresis followed by ethidium bromide staining.

Plasmid extraction

1. Culture in LB agar with proper antibiotics 37℃ 200 rpm for 14-16 hr.
2. Centrifuge cells at 15000 rpm for 10 sec and remove the supernatant.
3. Resuspend bacterial pellet.
4. Add 100µl solution l , vortex and react in 37℃ for 8 min.
5. Add 200µl solution ll for 3 mins.
6. Add 150µl solution lll and put on ice for 3-5 min. for reaction.
7. Centrifuge at 12000 rpm for 5 min.
8. Transfer 450µl supernatant into a new microcentrifuge tube.
9. Add 450µl Phenol/Chloroform, mix thoroughly by inverting several times.
10. Centrifuge at 12000 rpm for 5 min.
11. Transfer 320µl supernatant into a new microcentrifuge tube.
12. Add 900µl 100% Ethanol.
13. Centrifuge at 4℃ 12000 rpm for 5 min.
14. Wash the pellet with 900µl 70% Ethanol.
15. Centrifuge at 12000 rpm for 1 min to pellet down the DNA.
16. Discard the supernatant carefully.
17. Dry the pellet in a heat oven (60℃) for 10 min.
18. Add 33µl sterile water and resuspend the DNA pellet in 60℃.
19. Add 1µl RNase for 10-15min.
20. Evaluate amplified DNA by agarose gel electrophoresis followed by ethidium bromide staining.

Preparing Arabidopsis thaliana col-0 seedling

1. Use 75% ethanol sterilize the seeds for 30 second .
2. 1.5% NaClO to sterilize the seeds, constant shaking for 15 min.
3. Wash for 3-5 times and store in 4℃ to vernalize for 2-4 days.
4. Sow the seeds in 104 plug with BVB peat soil with vermiculite,the ratio of soil and vermiculite is 1:1.

Preparing rice (Oryza sativa var. Japonica Taiken 9) seedling

1. Sterilize the seeds with 1.5% NaClO for 15 min.
2. Soaked the seeds in water at 28C, constantly shaking for 3 day in darkness, the water is replaced every day.
3. After three days in darkness,sow the germinated seeds in 104 plug.

Preparing rice (Oryza sativa var. Japonica Taiken 9) seedling growth in MS medium

1. Remove the hull.
2. Sterilized the seed with 2% NaClO for 15 min and soaked in water at 28℃,constantly shaking for 1 day in darkness.
3. Sow in MS medium.

Preparing Murashige and Skoog medium

1. sucrose 1%
2. MES powder 0.1%
3. MS powder 4.43g/L
4. Adjust pH value to 5.8 by KOH
5. Gellan Gum Powder 0.3%
6. Moist heat sterilize.

Inoculation

1. Activate strain within 5ml LB medium containing 1/1000 Kanamycin under 37⁰C and constant shaking speed 150rpm overnight
2. Subculture in flask within LB medium under 37⁰C and constant shaking speed 150rpm until log phase.
3. Pellet down the strain with a centrifuge machine under 3000 xg at RT for 10 min, replace the supernatant with sterilised water and adjust final OD600 value to 1 as ‘inoculant’.
4. Filter the supernatant with .22 filter, add sterilised water and adjust final OD600 value to 1 as “sup”.
5. Take 20ul sup. on plate for control.
6. Inoculate 3 weeks old Arabidopsis thaliana Col-0,10ml for each plug.

Saccharified straw liquid inoculation

1. Activate strain within 5ml LB medium containing 1/1000 kanamycin under 37⁰C and constant shaking speed 150rpm overnight.
2. Subculture the strain in flask within saccharified straw liquid under 37⁰C and constant shaking speed 150rpm until log phase.
3. Adjust OD600 value to 1 ,pellet down the strain with centrifuge machine under 5000 xg for 10 min,filtr the supernatant with .22 filter.
4. Supernatant diluted 0X,100X,200X,500X times.

Re-isolation of Bacillus subtilis RM125/PQQ in plant

1. Soak in 75% ethanol for 30 seconds and in 0.6% NaClO with constant shaking for 12 min to sterilize the tissue.
2. Rinse by sterile water for five times and drip the final washing water on L.B. agar, then incubated at 37°C for 16 hours to ensure the sterilization effectiveness.
3. Homogenize in a sterile mortar pestle and dilute to 10-1, 10-2, 10-3 and 10-4 with sterile water.
4. 20-μL dilutions were dripped on the surface of solid media and incubated at 37°C for 16 hours.

Measure chlorophyll concentration

1. Soak sample in DMF and store in -4⁰C one day in darkness to extract the chlorophyll.
2. Use a spectrophotometer to measure the chlorophyll concentration.
3. The formula is as follows:
   
   Concentration (ug/mL)
   Ca=11.65*A664-2.69*A647
   Cb=20.81*A647-4.53*A664
   Cx+c=(1000*A480-0.89*Ca-52.02*Cb)/245
   
   Content (ug/g)
   X*(V/W)
   X: Chlorophyll concentration from the equation
   V: Volume of extraction solvent
   W: Weight of extraction sample
   

Data analysis

1. Record the phenotype in a manual way.
2. Use Shapiro-Wilk test for normal distribution,Levene’s test for homoscedasticity.
3. If the data conforms to normal distribution and homoscedasticity,statistical analyses of variance were performed using one-way ANOVA employing a post hoc Tukey-Kramer test. If the data didn’t conforms to normal distribution and homoscedasticity,statistical analyses of variance were performed using Kruskal-Wallis test employing a post hoc DUNN’s test. p-value < 0.05 was considered to be a significant effect.

• nchutaichung2021@gmail.com
• NCHU iGEM Team
• igem_nchu_taichung
• NCHU_Taichung
• NCHU, Taichung, Taiwan