Team:NNU-China/Project/Experiment
1. Construction of the library of BL21 (DE3)-derived variant strains
Materials & Apparatus
1. LB medium with 50 μg/mL kanamycin
2. LB medium with 50 μg/mL spectinomycin
3. 10% glycerin
4. 0.1M CaCl2
5. 0.1M CaCl2 with 10% glycerol
6. Centrifuge
7. Electric-heated thermostatic water bath
8. Electric Rotor Cup
9. MicroPulser electroporator
1.1 CRISPR
Before Experiment
1. 10μL bacteria solution was inoculated into 5mL LB liquid mediums at 37 °C, 200 rpm and last 12-16h.
2. Pellet 1.5mL bacteria solution from step 1 in a clean 2mL microcentrifuge tube by centrifugation at 4,000 rpm for 10 minute at 4℃ temperature. Decant or aspirate medium and discard.
3. Add 1mL 0.1M CaCl2, pipet up and down slightly to mix thoroughly, put the centrifuge tube on ice and let it stand for 30 min. And centrifuge at 4,000rpm for 10 minute at 4℃ temperature. Decant or aspirate medium and discard.
4. Add 100μL 0.1M CaCl2 with 10% glycerol, pipet up and down slightly to mix thoroughly.
5. Add 100-200 ng/μl pCas plasmid which contains cas9 gene into centrifuge .
6. Heat for 90S in a 42℃ water bath.
7. Add 600μL LB liquid medium, and incubate at 37 °C, 200 rpm for 1h.
8. Place the centrifuge tube in the centrifuge at 4,000 rpm for 3 minutes to collect the bacterial cells, discard part of the supernatant, mix the bacterial liquid and spread it on the LB solid plate with Kan.
9. Select the single bacteria on the LB solid plate, insert it into the LB liquid medium and cultivate for at least 12-16h.
Experiment
1. 10μL bacteria which contains pCas plasmid solution and 5μL kanamycin were inoculated into 5mL LB liquid mediums at 37 °C, 200 rpm and last 12-16h.
2. Pellet 1.5mL bacteria solution from step 1 in a clean 2mL microcentrifuge tube by centrifugation at 4,000 rpm for 10 minute at 4℃ temperature. Decant or aspirate medium and discard.
3. Add 1mL 10% glycerin, pipet up and down slightly to mix thoroughly. And centrifuge at 4,000rpm for 10 minute at 4℃ temperature. Decant or aspirate medium and discard.
4. Repeat step 3 once.
5. Add 100 μl 10% glycerin, and add 700 ng/μl pTarget plasmid which contains sgRNA scaffold into centrifuge.
6. Transfer all the bacteria liquid into the electro-rotor cup that has been standing on ice.
7. Electric shock under the conditions of 1.85kV, 200Ohm, 25μF.
8. Add 800μL LB liquid medium, and incubate at 37 °C, 200 rpm for 1h.
9. Place the centrifuge tube in the centrifuge at 4,000 rpm for 3 minutes to collect the bacterial cells, discard part of the supernatant, mix the bacterial liquid and spread it on the LB solid plate with Kan and Spec.
10. Single colonies were randomly selected for sequencing after plasmid loss.
11. Calculate the proportion of theoretical library = number of variants/theoretical value.
12. All single bacteria will be cultured in the same LB liquid medium.
1.2 CBE
Before Experiment
1. Use CRISPR editing technology to replace the RBS sequence on the BL21(DE3) genome with GGGGGGGG, named BL21(DE3)-8G.
2. 10μL BL21 (DE3)-8G bacteria solution was inoculated into 5mL LB liquid mediums at 37 °C, 200 rpm and last 12-16h.
3. Pellet 1.5mL bacteria solution from step 1 in a clean 2mL microcentrifuge tube by centrifugation at 4,000 rpm for 10 minute at 4℃ temperature. Decant or aspirate medium and discard.
4. Add 1mL 0.1M CaCl2, pipet up and down slightly to mix thoroughly, put the centrifuge tube on ice and let it stand for 30 min. And centrifuge at 4,000rpm for 10 minute at 4℃ temperature. Decant or aspirate medium and discard.
5. Add 100μL 0.1M CaCl2 with 10% glycerol, pipet up and down slightly to mix thoroughly.
6. Add 100-200 ng/μl pdCas-CDA-UGI-LVA plasmid into centrifuge.
7. Heat for 90S in a 42℃ water bath.
8. Add 600μL LB liquid medium, and incubate at 37 °C, 200 rpm for 1h.
9. Place the centrifuge tube in the centrifuge at 4,000 rpm for 3 minutes to collect the bacterial cells, discard part of the supernatant, mix the bacterial liquid and spread it on the LB solid plate with Kan.
10. Select the single bacteria on the LB solid plate, insert it into the LB liquid medium and cultivate for at least 12-16h.
Experiment
1. 10μL bacteria which contains pdCas-CDA-UGI-LVA plasmid solution and 5μL kanamycin were inoculated into 5mL LB liquid mediums at 37 °C, 200 rpm and last 12-16h.
2. Pellet 1.5mL bacteria solution from step 1 in a clean 2mL microcentrifuge tube by centrifugation at 4,000 rpm for 10 minute at 4℃ temperature. Decant or aspirate medium and discard.
3. Add 1mL 10% glycerin, pipet up and down slightly to mix thoroughly. And centrifuge at 4,000rpm for 10 minute at 4℃ temperature. Decant or aspirate medium and discard.
4. Repeat step 3 once.
5. Add 100 μl 10% glycerin, and add 700 ng/μl pTargetS pasmid which contains double sgRNA scaffolds into centrifuge.
6. Transfer all the bacteria liquid into the electro-rotor cup that has been standing on ice.
7. Electric shock under the conditions of 1.85kV, 200Ohm, 25μF.
8. Add 800μL LB liquid medium, and incubate at 37 °C, 200 rpm for 1h.
9. Place the centrifuge tube in the centrifuge at 4,000 rpm for 3 minutes to collect the bacterial cells, discard part of the supernatant, mix the bacterial liquid and spread it on the LB solid plate with Kan and Spec.
10. Single colonies were randomly selected for sequencing after plasmid loss.
11. Calculate the proportion of theoretical library = number of variants/theoretical value.
2. Fluorescence intensity measurement to screening high-yield strains
Material&Apparatus
1. Luria Bertani (LB) medium with 50 μg/mL kanamycin (Kan)
2. Terrific Broth (TB) medium with 50 μg/mL Kan
3. Isopropyl β‐D‐1‐thiogalactopyranoside (IPTG)
4. Shaker of 15mL tube or 250mL shake flask
5. 96-well ELISA plate of transparent or black
6. Enzyme-labeled instrument
Before Experiment
1. Single colonies and 0.8μL kanamycin were added into 800μL LB liquid mediums at 37 °C, 1200 rpm and last 12h in the 96-well transparent microplate.
Experiment
1. 10μL bacteria solution and 0.8μL kanamycin were added into 800μL TB liquid mediums at 37 °C, 1200 rpm and last 3h in the 96-well transparent microplate.
2. 10μL IPTG (0.1M) was added into above TB liquid mediums and continued to shake at 28°C, 1200 rpm and last 24h.
3. The samples of 200 μL were transferred into the 96-well transparent microplate for OD600 measurements.
4. The samples of 200 μL were transferred into the 96-well transparent microplate for GFP fluorescence intensity. The fluorescence intensity was measured at 485 nm excitation wavelength and 520 nm emission wavelength.
5. Measured the fluorescence intensity per OD600 to screening high-yield strains.
3. Fluorescence intensity measurement of fermentation broth
Material&Apparatus
1. Luria Bertani (LB) medium with 50 μg/mL kanamycin (Kan)
2. Terrific Broth (TB) medium with 50 μg/mL Kan
3. Isopropyl β‐D‐1‐thiogalactopyranoside (IPTG)
4. PBS buffer
5. Shaker of 15mL tube or 250mL shake flask
6. 96-well ELISA plate of transparent or black
7. Enzyme-labeled instrument
Before Experiment
1. Single colonies and 5μL kanamycin were added into 5mL LB liquid mediums at 37 °C, 220 rpm and last 12-16h.
Experiment
1. 200μL bacteria solution and 30μL kanamycin were added into 30mL TB liquid mediums at 37 °C, 220 rpm and last 3h.
2. 9μL IPTG(1M) was added into above TB liquid mediums and continued to shake at 28°C, 220 rpm and last 24h.
3. 10mL of 24h fermentation broth was collected by centrifugation at 10,000g for 10min.
4. Add 10mL PBS buffer, pipet up and down to mix thoroughly, and then disrupted using an ultrasonic cell disruptor (operating for 5 s, pausing for 5 s, 60 times).
5. The cell lysate was centrifuged at 4°C, 10,000g for 10 min, and the supernatant contained proteins of AMP-eGFP.
6. The samples of 200 μL were transferred into the 96-well transparent microplate for OD600 measurements.
7. The samples of 200 μL were transferred into the 96-well transparent microplate for GFP fluorescence intensity. The fluorescence intensity was measured at 485 nm excitation wavelength and 520 nm emission wavelength.
8. Measured the fluorescence intensity per OD600 to assess AMPs expression levels.
4. SDS-PAGE
Material&Apparatus
1. Luria Bertani (LB) medium with 50 μg/mL kanamycin (Kan)
2. Terrific Broth (TB) medium with 50 μg/mL Kan
3. Isopropyl β‐D‐1‐thiogalactopyranoside (IPTG)
4. Shaker of 15mL tube or 250mL shake flask
5. PBS buffer
6. 96-well ELISA plate of transparent or black
7. Enzyme-labeled instrument
8. Bio-Rad® SDS-PAGE Gel preparation kit
9. Tris Powder
10. SDS
11. APS
12. TEMED
13. SDS-PAGE Sample Loading Buffer,10X (produced by Sangon Biotech®)
14. Glacial acetic acid
15. Protein samples
16. Protein marker 180kDa (produced by Sangon Biotech®)
17. 50mL centrifuge tube
18. Electrophoresis power supply
19. Microwave oven
Before Experiment
1. Single colonies and 5μL kanamycin were added into 5mL LB liquid mediums at 37 °C, 220 rpm and last 12-16h.
Experiment
1. 200μL bacteria solution and 30μL kanamycin were added into 30mL TB liquid mediums at 37 °C, 220 rpm and last 3h.
2. 9μL IPTG was added into above TB liquid mediums and continued to shake at 28°C, 220 rpm and last 24h.
3. 10mL of 24h fermentation broth was collected by centrifugation at 10,000g for 10min.
4. Add 10mL PBS buffer, pipet up and down to mix thoroughly, and then disrupted using an ultrasonic cell disruptor (operating for 5 s, pausing for 5 s, 60 times).
5. The cell lysate was centrifuged at 4°C, 10,000g for 10 min, and the supernatant contained soluble proteins.
6. 50μl of the supernatant was mixed with 50μl of 10x loading buffer, and heated in a metal bath at 100°C for 10min.
7. The protein samples corresponding to 0.15OD were added to the protein gel wells, and separated at 80 V until the die reached the end of the gel.
5. Antibacterial experiment
Material&Apparatus
1. Luria Bertani (LB) medium
2. Escherichia coli MG1655
3. Aseptic water
4. Agar
5. Pipettor
6. tweezer
7. Clean bench
8. Oxford Cup
9. 37℃ incubator
Before Experiment
1. Single colony was added into 5mL LB liquid mediums at 37 °C, 220 rpm and last 12-16h.
Experiment
1. The 10mL water agar medium were poured into the plates and condensed.
2. The 10mL bacteria solution was added to the LB medium at 45-50 ℃ and poured into the water agar medium (5mL).
3. Six oxford cups were placed with disinfection tweezer, one of which added sterile water as control, and the others added different concentrations of AMPs.
4. After 12 hours, the diameter of bacteriostatic zones were measured to evaluate the antibacterial activity of AMPs.
