Experiment

Duke Kunshan University DKU iGEM Team 2021

Experiments:

LB recipe, Making competent E. coli, Bacterial transformation, IPTG induction for protein expression, Purification of His-tagged protein with BeyoGold™ His-tag Purification Kit, SDS-page gel electrophoresis, SDS-page gel rapid staining, Ethanol precipitation, Agarose gel electrophoresis, CRISPR to prepare gene knock-out strain of E.coli for exosome synthesis.

LB recipe:

10g tryptone, 10g NaCl, 5g Yeast extract into 1L water. Autoclave. If want to make LB agar, add 12g Agar-Agar per liter and autoclave.

Making competent E. coli:

1 Prepare autoclaved LB media, 100mM CaCl2, 100mM MgCl2, 85 mM CaCl2, 15% glycerol v/v. Chill at 4 °C.
2 Incubate the bacteria until OD600=0.4. Put cells on ice.
3 Fill the 1.5 mL microcentrifuge tube with culture. Harvest cells by centrifuging at 4000g for 10 min at 4 °C.
4 Decant the supernatant.
5 Repeat step 3. Resuspend pellet in 1mL of ice-cold MgCl2.
6 Harvest the cells by centrifuging at 3000g for 10 minutes at 4°C.
7 ecant the supernatant. Resuspend the pellet in 1.5 mL of ice-cold CaCl2. Keep on ice for 10 minutes. Set 1.5 mL microcentrifuge tubes on ice.
8 Harvest the cells by centrifuging at 3000g for 10 minutes at 4°C.
9 Decant the supernatant and resuspend the pellet in 0.5mL of ice-cold 85 mM CaCl2, 15% glycerol.
10 Harvest the cells by centrifuging at 2000g for 10 minutes at 4°C.
11 Decant the supernatant and resuspend the pellet in 1 mL of ice-cold 85 mMCaCl2, 15% glycerol.
12 Snap freeze with liquid nitrogen. Store frozen cells in the -80°C freezer.

Bacterial transformation:

Take competent cells on ice. Wait for 20 min.
Mix1-5 μL (10 pg-100 ng) plasmid with 50 μL of competent cells. Flick the tube.
Incubate on ice for 30 min.
Heat shock at 42 °C for 45 s.
Put back on the ice for 2 min.
Add 250 μL of LB to the bacteria, shake at 37 °C for 45 min.
Spread onto LB agar plates with specific selective stress (antibiotics that the plasmid provides resistance to).

IPTG induction for protein expression

Pick a colony to 3mL of LB with antibiotics that the bacteria is resistant to.
Shake at 37 °C degrees overnight. Add 1/30 volume of overnight culture to fresh LB with antibiotics. Incubate until OD600 reaches 0.6.
Take out some culture as a control group to incubate separately. Add IPTG to the final concentration of 0.1-1mM.
Shake at 37 °C for 6 h or 18 °C overnight if the protein is toxic.
Centrifuge at 15000g to harvest the cells for later processes.

Purification of His-tagged protein with BeyoGold™ His-tag Purification Kit*
*Modified from BeyoGold™ His-tag Purification Kit protocol (https://www.beyotime.com/product/P2226.htm)

A. Small amount extraction.

Thaw bacteria on ice.
Resuspend the bacteria in Beyotime Non-denature lysis buffer: 100 μL per 1mL induction culture. Add 1 μL of 1M PMSF. Add lysozyme to 1mg/mL. Vortex briefly and chill on ice for 30 min.
Vortex and centrifuge at 15000g for 10 min at 4 °C. Collect some cell lysate (CL). Transfer all the remaining supernatants to another tube.
Add 20 μL of BeyoGold™ His-tag Purification Resin to the new tube, shake at 4 °C for 30 min.
Centrifuge at 10000g for 30 s at 4 °C. Collect the supernatant or Flow-through (FT).
Add 40 μL of non-denature washing buffer. Resuspend. Centrifuge at 10000g for 30 s at 4 °C. Decant supernatant.
Repeat step 7.
Add 20 μL non-denature elution buffer. Resuspend. Centrifuge at 10000g for 30 s at 4 °C. Collect the supernatant as elution (E).

B. Large amount extraction.

Thaw bacteria on ice.
Resuspend the bacteria in Beyotime Non-denature lysis buffer: 4 mL per gram of pellet. Add PMSF to 1mM.
Supersonic disruption: 300W, 50 min, 4s disruption and 6s rest per cycle. All performed on ice.
Centrifuge at 15000g for 20 min. Transfer supernatants to new tubes. Collect some as cell lysate (CL).
Add 2 mL BeyoGold™ His-tag Purification Resin to the supernatant. Shake at 4 °C overnight.
Load the mixture to an empty column. Collect the Flow-through (FT).
Wash the column with 1 mL washing buffer 5 times.
Elute with elution buffer for 4 times, collect eluted solution as Elution1 (E1), Elution2+3 (E23), and Elution4 (E4).

SDS-page gel electrophoresis:

Mix 20 μL of the sample with 5 μL 5X Protein Loading buffer: 10% SDS, 500mM DTT, 50% Glycerol, 250mM Tris-HCL and 0.5% bromophenol blue dye, PH6.8.
Place in 90-degree water for 1 minute.
Load the mixture to the well of an SDS-page gel.
Run at 30V, 300mA for 30 minutes for stacking purposes.
Run at 120V, 300mA until the bromophenol blue reaches the bottom of the gel.
Take the gel out.

SDS-page gel rapid staining:

After electrophoresis, the gel is washed off the glass plates with water and soaked in gel fixing solution (30%(v/v) Ethanol, 5%(v/v) Phosphoric acid in Milli-Q water) for 1hr.
Wash with water 2-3 times.
Cover the gel with the rapid staining solution (0.02%(w/v) Coomassie Brilliant Blue G250, 5%(v/v) Phosphoric acid, 20%(v/v) Ethanol, 5%(w/v) Ammonium sulfate in Milli-Q water, filtered before use). Shake at room temperature overnight.
Destain with DI water for 6 h.
The gel can be stored on DI water for several weeks.

Ethanol Precipitation

1. Hold all the reagents and DNA sample in ice box before precipitation.
2. Add 0.1 volume of NaAc and 2.5 volume of 2.5 absolute ethanol. Mix by vortexing.
3. (Optional) add 2 µL of glycogen or polyacrylamide and vortex.
4. Let stand in -20 ˚C refrigerator for over 30 minutes. Extend the freezing time or use a -80 ˚C refrigerator will give more the precipitation.
5. Centrifuge at full speed at 4 ˚C for over 15 minutes.
6. Remove the supernatant by pipetting or carefully pouring.
7. Add 200 µL of the 70% ethanol to wash the pellet.
8. Centrifuge at full speed at 4 ˚C for over 10 minutes.
9. Remove the supernatant carefully. Open the tube for ethanol to evaporate.
10. Resuspend the pellet in 40 µL of MilliQ water or buffer.

Agarose gel electrophoresis*

*Used nucleic acid dye from SolarBio (https://www.solarbio.com/pdf/2020-shsj/G5560.pdf).
Weigh 1g of agarose in 100 mL TAE buffer. Microwave until fully dissolved.
After cooled down to 50 °C, add 10 μL SolarGelRed Nucleic Acid Gel Stain (10000×). Mix.
Pour the mixture into the mold with combs firstly placed on it.
After solidified, take off the comb.
Soak the gel in the TAE buffer in the gel running track. Mix samples with the proper amount of loading buffer and load them into the wells (usually 10-15μL).
Run at 120V, 300mA for 50 min.
Take a photo of the gel under UV light in any kind of gel imaging system.

CRISPR to prepare gene knock-out strain of E.coli for exosome synthesis:

Transform Plasmid A (pCas) into bacteria through electroporation and select on plate containing kanamycin at 30 ℃. Select positive clones to prepare competent cells, and add 10mM arabine 1 hour before centrifugal collection to induce the expression of RED.
Transform Plasmid B (pTargetT) with homologous sequence into bacteria through electroporation and select positive clones with kanamycin and streptomycin at 30 ℃.
Do overnight culture.
Inoculate Positive clones in LB liquid (Kan resistance) and cultured with 0.5mm IPTG for 8-20 hours at 30 ℃.
Verify the elimination of plasmid B (pTargetF).
Culture the bacteria at 37 ° C to eliminate plasmids A (pCas)
Verify the gene know-out by Sanger sequencing.
For the gene knock-out strains, culture them in LB media at 37° C before harvest.
Purify the exosome via ultracentrifugation
Examine the quality and the yield through flow-cytometry and fluorescent staining.

References:

Beyotime. BeyoGold™ His-tag Purification Kit. https://www.beyotime.com/product/P2229S.htm NEB. (2014). Single-temperature Double Digest. https://www.protocols.io/view/Single-temperature-Double-Digest-imsuj5 Solarbio. SolarGelRed Nucleic Acid Gel Stain (10000×). Retrieved October, 12 from https://www.solarbio.com/pdf/2020-shsj/G5560.pdf

Acknowledgments:

Special thanks to Ziqin Wang and Yang Yu for summarizing the protocols from their lab experience as student workers in the Molecular Biology Synear Lab of Duke Kunshan University.

Experiment Notebook

The following link is our daily experiment notebook.

https://docs.google.com/document/d/1SsGdR3X8mdSPIwK2Y9mHJxLnnts9W6B3/edit?usp=sharing&ouid=100362513723710408555&rtpof=true&sd=true

Team:DKU/Experiment

DKU igem