Experiments

gDNA purification

Materials:

SETLR buffer	SETL	Sucrose	2g
20% Sucrose		500mM EDTA	0.4ml
20 mM EDTA		1M Tris(pH7.9)	0.5ml
50 mM Tris-HCl pH7.9		Lysozyme	0.02g
0.2% Lysozyme		dil. w/ MQ to 10mL
100 μg/mL RNaseA
Store at 4℃	SETLR	SETL buffer	10ml
		100 mg/mL RNaseA	10ul

Procedure:

Preparation: culture bacteria overnight in a shaking incubator.
1. Centrifuge 1 mL of culture in 1.5 mL centrifuge tube at 10,000 rpm for 1 minute to pellet the cells. Remove the supernatant.
2. Resuspend the cells thoroughly in 100 µl of SETLR buffer.
3. Incubate at 37℃, for 20 min.
4. Add 10 μL of 10% SDS.
5. Mix thoroughly by inverting the tube.
6. Flash tube.
7. Add 400 μL of TE buffer.
8. Mix thoroughly by inverting the tube.
9. Flash tube.
10. Add 0.26 g of ammonium sulfate.
11. Mix thoroughly by inverting the tube.
12. Stand on ice for 15 min.
13. Centrifuge at 15,000 rpm for 15 minutes at 4℃.
14. Collect supernatant to a new 1.5 mL tube.
15. Add 300 μL of isopropanol.
16. Mix thoroughly by inverting the tube.
17. Confirm a visible thread-like particle.
18. Centrifuge at 15,000 rpm for 10 minutes at 4℃.
19. Discard supernatants.
20. Add 900 μL of 70% Ethanol.
21. Mix thoroughly by inverting the tube.
22. Centrifuge at 15,000 rpm for 3 minutes at 4℃.
23. Discard supernatants.
24. Add 900 μL of 70% Ethanol.
25. Mix thoroughly by inverting the tube.
26. Centrifuge at 15,000 rpm for 3 minutes at 4℃.
27. Discard supernatants.
28. Air dry for 10 min at 37℃ incubator.
29. Dissolve the pellet with 100 μL of TE buffer.

Inoculation

Procedure:

1. Prepare eight 50mL Falcon tubes
2. Pour in 5mL of liquid LB medium to each tube
3. Use a toothpick to pick up bacteria. Label samples.

Glycerol Freeze Stock of bacterial strains

Procedure:

1. Measure OD600 value for the O/N culture of the strains. (1/10 dil. with LB medium)
2. Add 500 μL of O/N culture to 250 μL of autoclaved 80% Glycerol
3. Store at -80℃ (in deep freezer)

Buffer & Gel preparation (for PCR)

TAE-EtBr buffer

1. dilute 160mL of 10x TAE to 1.6L
2. Add 80mL of EtBr ※Avoid EtBr being under direct light

2% TAE-EtBr agarose gel

1. Mix 50ml of TAE-EtBr + 1g agar
2. Microwave until solution becomes clear
3. Aliquot into four molds

0.8% TAE-EtBr agarose gel

1. Mix 75ml of TAE-EtBr + 0.6g agar
2. Microwave until solution becomes clear
3. Aliquot into four molds

PCR

Electrophoresis

PCR Clean-up (Column Purification)

Procedure:

1. adjust DNA binding solution (200ul buffer NTI + 100ul sample)
2. place NucleoSpin Gel and PCR Clean-up Column into a collection tube (2ml) a load up to 700 ul of sample
3. centrifuge for 30s at 11,000 f g
4. discard flow-through and place column back into collection tube (load remaining sample if necessary and repeat centrifuge step)
5. add 700ul buffer NT3 to the NucleoSpin Gel and PCR Clean-up column
6. centrifuge for 30s at 11,000 x g
7. discard flow-through and place the column back into the collection tube
*recommended: repeat various washing step (5-7) to minimize chaotropic salt carry-over and improve A260/A230 values
8.centrifuge for 2 min at 11,000 x g to remove buffer NT3 (make sure spin column doesn’t come in contact with the flow-through while removing it from the centrifuge and collection tube)
*Residual ethanol from Buffer NT3 might inhibit enzymatic reactions. Total removal of ethanol can be achieved by incubating the columns for 2–5 min at 70 °C prior to elution.
9. place the NucleoSpin Gel and PCR Clen-up column into a new 1.5ml microcentrifuge tube
10. add 30ul Buffer NE and incubate at room temperature (15-25°c) for 1 min
11. centrifuge for 1 min at 11,000 x g
*DNA recovery of larger fragments (> 1000 bp) can be increased by multiple elution steps with a fresh buffer, heating to 70 °C and incubation for 5 min.

Preculture of E. coli C600 to prepare competent cell

Procedure:

1. Pour in 5mL of LB liquid medium into a 50mL falcon tube
2. Go to the freezer and take a bit of the glycerol stock “GTstr.7” using a pipette tip, dissolve in to medium
3. Shake o.n.

Preparation of E. coli competent cell

Procedure:

1. Shake E. coli at 37 °C overnight in 5 ml of LB.
2. Add 0.4 ml of the overnight culture into 40 ml of LB in a 50 mL tube.
3. Monitor OD600. When OD600 of 0.35-0.4 is reached (about 120 min), chill the culture on ice. @innova
4. Collect cells by centrifugation at 8,000 rpm for 5 minutes at 4 °C. Discard the supernatant.
5. Resuspend the cells in 20 ml of ice-cold 50 mM CaCl2. Incubate the resuspended cells on ice for 20 minutes. Collect cells by centrifugation as in step 4.
6. Resuspend the cells in 3 ml of ice-cold 50 mM CaCl2 containing 10% glycerol.
7. Dispense the competent cells into aliquots of 100 μL, freeze in liquid nitrogen, and store at -80℃.

In-Fusion

Transformation into E. coli DH5α

Procedure:

1. Mix 25μl of E. coli DH5α competent cells with 1μL of plasmid DNA.
2. Put on ice for 10 min, incubate at 42℃ for 45 sec, put on ice for 2 min.
3. Add 500 μl of LB medium, put in RT for 5 min, aliquot into 2 LB plates each.
4. Shake & incubate at 37℃, O/N

Transformation of dimer plasmid into B. subtilis

Materials: (8/9 protocol doc)

Procedure:
Without freeze (protocol from Prof. Asai)

o.n. culture on LB plate already prepared yesterday
1. Innoculate the o.n. culture of B. subtilis in 5ml MDCH to OD600=0.1. Shake at 37℃
2. After 3-4 hours, when OD growth curve begins to flatten, add MD to match the MDCH volume of the moment. Culture of 30 more minutes
3. Aliquot the solutions by 200μl to 10ml test tubes. Add DNA, incubate for 1 hour
4. Centrifuge the cells, 10,000rpm 1 min. Remove 100μl of supernatant
5. Resuspend cells. Plate onto a selective medium
6. Culture for 30℃ o.n.

With freeze (altered the protocol from Prof. Asai)

1. Innoculate the o.n. culture of B. subtilis in 25ml MDCH to OD600=0.1. Shake at 37℃
2. After 3-4 hours, when OD growth curve begins to flatten, add MD to match the MDCH volume of the moment. Culture of 30 more minutes
3. Centrifuge the cells, 8,000 rpm 5 min at 25℃. Preserve 4 ml of supernatant, and throw away the rest.
4. Resuspend cells with 4 ml of preserved supernatant
5. add 0.75ml of 80% glycerol
6. Aliquot the solutions by 200μl to 1.5 ml tubes.
7. Freeze with liquid nitrogen, store in -80℃ freezer
8. Thaw 4 of the solutions. Add DNA/plasmid as listed below, and transfer each liquid to 10ml test tubes. Incubate for 20 min
9. Add 1 ml of LB liquid medium. Shake at 37 degrees for 45 min.
10. Spread 100μl to selective LB medium (LB+sp+cm plate)
11. Transfer all remaining culture to 1.5 ml tube. Centrifuge 10,000 rpm for 1 min. Leave 100μl and remove the rest of the supernatant.
12. Resuspend cells with the 100μl of culture that was preserved.
13. Culture for 30℃ o.n.

Transformation into E. coli C600

1. Mix 200 μl of E. coli C600 competent cell with 3μL of plasmid DNA
2. Put on ice for 10 min, incubate at 42℃ for 45 sec, put on ice for 2 min.
3. Add 500 μl of LB medium, put in RT for 5 min, aliquot into 2 LB plates each, one for 25 μl and one for the rest.
4. Shake & incubate at 37℃, O/N

Miniprep (with QIAprep Spin Miniprep Kit)

Materials:

Procedure:

1. Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C).
2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube (1.5ml tube).
3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes blue (when using LyseBlue reagent).
4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the solution will turn colorless.
5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
6. Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting.Centrifuge for 30–60 s and discard the flow-through.
7. Wash the QIAprep spin column by adding 0.5 ml Buffer PB. Centrifuge for 30–60 s and discard the flow-through.
8. Wash the QIAprep spin column by adding 0.75 ml Buffer PE. Centrifuge for 30–60 s and discard the flow-through.
9. Centrifuge for 1 min to remove residual wash buffer.
10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) to the center of the QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

Conjugation

Procedure:

Preculture the Donor and the Recipient strain in 5 mL of LB medium overnight at 37 degrees C with shaking.
Inoculate the overnight culture to 5 mL of fresh LB medium (if necessary add antibiotics) to give OD = 0.1.
Shake at 37 degrees C for 3 hours.
Monitor the OD values.
Centrifuge the culture containing 1 x 10^9 cells (OD = 1 gives 8 x 10^8 cells).
Discard the supernatant and re-suspend the cells to 1 mL of fresh LB medium (***DO NOT STIR WITH A VORTEX MIXER)
Centrifuge the cells and discard the supernatant.
Re-suspend the cells to 0.1 mL of fresh LB medium.
Mix each of the 0.1 mL of Donor and Recipient in a 1.5 mL tube.
Stand for 30 min at room temperature.
Take 50 μL of the culture and spread onto a selection plate.
Incubate at 37 degrees C overnight.

Milk plate assay

Materials (per plate):

Reagent	Volume / Weight	Concentration
agarose solution	14.3ml	2%(final 1.1%)	0.286g agarose + MQ 14.3ml
skim milk	0.05g	final 0.2%
sodium phosphate	10.7ml	0.1M(final 42mM)

Procedure:

1. mix sodium phosphate solution w/ skim milk at room temperature
2. dissolve agarose into MQ w/microwave and cool to 50-60°c
3. add skim milk solution and mix quickly
4. solidify for 1 hour in petri dish @room temperature
5. Use a large orifice pipette tip to make holes inside the fibrin plates. Let it dry.
6. Calculate and dilute the cell culture to OD=0.5 using the culture medium.
7. Add 4 μL of the resulting solution to each of the holes inside the fibrin plates.
8. Incubate at 37 degrees C overnight.

Fibrin plate assay

Materials (per plate):

Reagent	Volume / Weight	Concentration
fibrinogen	10.4ml	0.8mg/ml	in 0.1M sodium phosphate (final 42mM)
agarose	13.9ml	2% (final 1%)
thrombin	0.69ml	7.5U/ml

Thrombin preparation:

1. mix 0.8mg thrombin (solid) with 100 ul of 1% BSA and 900 ul milli Q
2. mix 52ul of thrombin solution with 638ul milli Q

Procedure:

NB, LB culture	Natto bean suspension liquid
1. add 0.8mg/ml fibrinogen in 15 ml of sodium phosphate solution (0.1M)
2. dissolve agarose into MQ w/microwave and cool to 50-60°c
3. leave in 45°c, 15min
4. add thrombin solution and mix quickly
5. pour into a petri dish
6. solidify for 1 hour in petri dish @room temperature
7. Use a large orifice pipette tip to make holes inside the fibrin plates. Let it dry.
8. Measure the OD of each cell culture.	8. Add 5mL of NB medium and one natto bean to 15 mL tubes.
	9. Vortex for 5 seconds.
	10. Transfer 1 mL of supernatant into 1.5 mL tubes. Measure the OD of each.
9(11). Calculate and dilute the cell culture to OD=0.5 using the culture medium.
10(12). Add 4 μL of the resulting solution to each of the holes inside the fibrin plates.
11(13). Incubate at 37 degrees C overnight.

Creating Natto using soybean and NK (black soybean, roasted soybean)

Procedure:

1. Wash off the dirt of the soybeans thoroughly with water
2. Place soybeans in a container with water covering the beans. Let it sit for 18 hours. *The soybeans should be around 2 times the size before after letting it sit in water
3. Culture NK overnight.
4. Place soybeans in two separate 50 mL tubes (soybean, black soybean).
5. Autoclave for 40 minutes at 120 degrees Celsius, then let sit for 10 minutes to cool.
6. Transfer the beans to Petri dishes.
7. Add 1 mL each of NK cultures to the corresponding petri dishes.
8. Culture overnight at 37 degrees Celsius.
9. Store at 4 degrees Celsius.