Experiments

Dilution of DNA fragments

DNA fragments stored at 20°C were thawed.

4μl of DNA fragments and 6 µl of TE buffer(pH 8.0) were mixed by pipetting in a 0.2 ml tube. The concentration was adjusted to 20 ng/µl.

https://static.igem.wiki/teams/4955/wiki/protocols/dilution-of-dna-fragments.pdf

Dilution of primer

Primers stored at -20°C were thawed.

TE buffer of ㏗8.0 stored in a refrigerator at 4°C was removed, and 5 µl of primers and 45 µl of buffer were mixed by pipetting in a 0.2 ml tube.

https://static.igem.wiki/teams/4955/wiki/protocols/dilution-of-primer.pdf

PCR

	Conc.	final	(µl)
Milli-Q H₂O			21.0
KOD One PCR Master Mix (TOYOBO)	2x	1x	25.0
DNA Template	1.0	0.0	1.0
Fwd Primer	10.0	0.3	1.5
Rev Primer	10.0	0.3	1.5
total			50.0

　↓

Thermal Cycler

98C	2min
98℃	10 sec	×25 cycle
(Tm -2~5C)℃	5sec
68℃	(5sec / kb)sec
12℃	∞

Overlap-PCR

	Conc.	final	(µl)
Milli-Q H₂O			4.5
KOD One PCR Master Mix (TOYOBO)	2x	1x	7.5
pgm fr1 (ng/ul)	20.0		1.0
pgm fr2 (ng/ul)	20.0		1.0
pgm fr3 (ng/ul)	20.0		1.0
			15.0

　↓

Thermal Cycler

98℃	10 sec	×15 cycle
55℃	5sec
68℃	30sec
12℃	∞

Electrophoresis (EtBr)

	(μl)
1.5x Stop K	2.0
	1.0
total	3.0

　↓

2.0μl：Apply to 1% Agarose, 0.5% TAE-EtBr gel

Electrophoresis at 100V, 15min

Electrophoresis (SYBR Green)

	(μl)
1.5x Stop K	2.0
	1.0
total	3.0

　↓

2.0μl：Apply to 0.5% Agarose, gel

Electrophoresis at 100V, 15min

　↓

1h staining in SYBR Green

Dpn1 processing

1 µl of SpeedCut Dpn I (LABTAS) enzyme stored at -20°C and 50 µl of PCR products were mixed and reacted at 37°C for 15 min.

https://static.igem.wiki/teams/4955/wiki/protocols/dpn1-processing.pdf

Preparation of LB Broth

25 g of LB Broth, Miller (nacalai tesque, product number (catalog number):20068-75) was suspended in 1 L deionized water and autoclaved at 121°C for 15 minutes.

Storage was done at room temperature.

https://static.igem.wiki/teams/4955/wiki/protocols/protocol-preparation-of-lb-medium.pdf

Reference

https://www.thermofisher.com/order/catalog/product/jp/ja/12780052

Transformation to DH5α

Quickly thaw Champion™ Competent Cell(SMOBIO) by hand (body temperature), warm

water bath or running buffer for approximately 10-20 seconds until 1/3-1/2 volume is thawed.

Add DNA to less than 10% of the competent cell volume.

Vortex for 10 seconds or tap the tube with your finger to mix well.

Incubate on ice for 0-10 minutes. (Incubation on ice slightly increases efficiency.)

Heat shock competent cells at 42°C for 45-90 seconds.

Incubate on ice for 1-5 minutes.

Add 900µL of LB or SOC medium to competent cells and incubate at 37°C for 30-60 minutes at 200 rpm with shaking.

Plate on pre-warmed (room temperature to 37°C), selective LB agar plates (LB + antibiotics).

Incubate the plate at 37°C until the colonies are suitable for analysis.

https://static.igem.wiki/teams/4955/wiki/protocols/transformation-to-dh5a.pdf

Transformation of BL21

Thaw a tube of BL21(DE3) Competent _E. coli _cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.

Add 1–5 µl containing 1 pg–100 ng of plasmid DNA to the cell mixture.

Carefully flick the tube 4–5 times to mix cells and DNA.

Place the mixture on ice for 30 minutes. Do not mix.

Heat shock at exactly 42°C for exactly 10 seconds. Do not mix.

Place on ice for 5 minutes. Do not mix. Pipette 950 µl of room temperature SOC into the mixture.

Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.

Warm selection plates to 37°C.

Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.

Spread 50–100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24–36 hours or at 25°C for 48 hours.

https://static.igem.wiki/teams/4955/wiki/protocols/transformation-of-bl21.pdf

Agar medium preparation

The lid of LBaga (common in the lab) prepared in 250 ml medium bottles was loosened and heated in a microwave oven at 600 W for 1 minute, then at 600 W for 30 seconds each until completely melted.

Running water was applied to the bottles to cool them to near human body temperature. Appropriate antibiotics were added. (The amount of antibiotics are shown below.)

20 ml was poured into a plastic petri dish and air bubbles were removed.

The lid of the petri dish was closed and the gel was left at room temperature for about 20 minutes until it hardened.

For storage after completion, the culture media prepared with the lid on the bottom side were wrapped together in saran wrap and placed in a refrigerator at 4°C.

Amount of antibiotic added

50 μl of Cb100μg/ml

125 μl of tet10μg/ml

100 μl of spec100μg/ml

100 μl of Gent7μg/ml

https://static.igem.wiki/teams/4955/wiki/protocols/agar-medium-preparation.pdf

Miniprep

TE buffer was pre-warmed to 70°C.

5ml of Culture was RT at 8000 rpm for 3min and the supernatant was discarded.

Pipetted 250µl of P1 reagent and transferred the contents to a 2 ml tube.

Add 250 μl of P2 reagent and mix 4~~6 times by inversion, then add 250 μl of N3 reagent and mix 4~~6 times by inversion.

After 2 min of standing in ice, centrifugation was performed at room temperature for 13 kprm 15 min.

The supernatant was added to the column, centrifuged at room temperature for 13kprm1min, and the flow-through was discarded.

500 μl of PB reagent was added, centrifuged at room temperature for 13 kprm30s, and the flow-through was discarded.

Add 750 PE reagent, centrifuge at room temperature for 13kprm1min, and discard the flow-through. Centrifugation and discarding the flow-through were performed twice.

The contents were transferred to a 1.5-ml tube and evaporated at room temperature for 5 min.

50 μl of warmed TE buffer was added, allowed to stand at room temperature for 1 min, and then centrifuged at 13 kprm 1 min at room temperature.

https://static.igem.wiki/teams/4955/wiki/protocols/miniprep.pdf

Ethanol precipitation

Step.1 Add 1/10 volume of 3M sodium acetate and 3x volume of 99.5% ethanol Step.2 Pipetting Step.3 Centrifuge at 15000 rpm for 10 min Step.4 Decantation Step.5 Add 500 μl of 70% ethanol stored at 4°C Step.6 Centrifuge for 5min without suspension Step.7 Discard the supernatant Step.8 Place in desiccators and wait for 10 min Step.9 Suspend in appropriate volume of milliQ

https://static.igem.wiki/teams/4955/wiki/protocols/ethanol-precipitation.pdf

Preparation of TB medium

47 g of Invitrogen™‘s Terrific Broth (product number (catalog number): 22711022) and 4 ml of glycerol were suspended in 1 L deionized water and autoclaved at 121°C for 15 minutes.

Storage was done at room temperature.

Reference

https://www.thermofisher.com/jp/ja/home/technical-resources/media-formulation.141.html

https://static.igem.wiki/teams/4955/wiki/protocols/preparation-of-tb-medium.pdf

Culture for substance production

Five mL of LB liquid medium was placed in a test tube and the appropriate amount of the

appropriate antibiotics were added.

Amount of antibiotic added

2.5 μl of Cb100μg/ml

6.25 μl of tet10μg/ml

5 μl of spec100μg/ml

5 μl of Gent7μg/ml

Bacteria were suspended in liquid medium and incubated at 37°C for 18-24 hours with

horizontal shaking as a pre-culture.

In the main culture, 1 ml of the pre-culture solution, 49 ml of TB liquid medium, and 50㎖ of

final were placed in a 300 ml flask and incubated at 25°C with horizontal shaking at 150 rpm until the OD value reached 8 to 12.

During this culture, 0.1 mM IPTG was added when the OD value reached 8, and induction

was performed.

Every 12 hours, 200 µl were sampled from 72~96 hours and stored at -20°C.

https://static.igem.wiki/teams/4955/wiki/protocols/culture-for-fluorescence-microscopy.pdf

OD measurement

110 μl of the liquid medium used in the material to be measured was placed in a plastic

cuvette, and the Blank was set.

The liquid medium in the cuvette was completely aspirated with an aspirator, and 110 μl of the material was added for measurement.

If the OD value exceeded 1.5, the direction in which the cuvette was placed in the analyzer

was changed by 90°. This would reduce the measured OD value by 1/5.

When the value still exceeded 1.5, the sample was appropriately diluted with liquid medium with the Blank set and mixed by pipetting before measurement.

When changing the sample to be measured or when terminating the measurement, the liquid in the cuvette was first completely absorbed with an aspirator and 80% ethanol was sprayed inside the cuvette.

Then, the ethanol in the cuvette was aspirated using an aspirator and rinsed with deionized water.

The deionized water remaining in the cuvette was aspirated with an aspirator.

https://static.igem.wiki/teams/4955/wiki/protocols/od-measurement.pdf

TSS

Step.1 LB 5ml + poke the bacteria with the appropriate antibiotic for each.

Step.2 Incubate to OD600=0.8 (37°C, 180 rpm)

Step.3 Transfer to a 1.5ml tube and centrifuge (5000g,1min)

Step.4 Suspend in 100 μl of room temperature TSS

Step 5 Add 2 μl of the plasmid you wish to insert and pipette again.

Step.5 on ice 15min

Step.6 42℃,45sec

Step.7 on ice 2 min

Step.8 Add 200 μl SOC

Step.9 Recovery incubation at 37°C for 60min.

Step.10 Centrifuge (5000g, 1min) and remove the medium.

Step.11 Resuspend in 50 μl of LB

Step.12 Sprinkle the appropriate amount on the appropriate plate.

https://static.igem.wiki/teams/4955/wiki/protocols/tss.pdf

Electroporation

Electroporation Competent cell preparation for electroporation

Step.1 Incubate the bacteria to be made into competent cells in 5 ml of LB overnight with

shaking (37°C, 180 rpm). Include appropriate antibiotics.

Step.2 Shake culture with 1ml of the culture medium of Step.1 and 99ml of LB until

OD600=0.4~0.5 (37°C, 180 rpm)

Step.3 on ice 15 min (all operations below are on ice)

Step.4 Dispense the culture medium into two 50 ml falcon tubes.

Step.5 Centrifuge (3000g , 5 min , 4°C), remove supernatant and add 40 ml 10% glycerol

(4°C) (20 ml added after suspension)

Step.6 Centrifuge (3000g , 5 min , 4°C), remove supernatant and add 10 ml cold 10%

glycerol (4°C)

Step.7 Centrifuge (3000g , 5 min , 4°C), remove supernatant and add 5 ml cold 10% glycerol (4°C)

Step.8 Centrifuge (3000g , 5 min , 4°C), remove supernatant and add 200 μl of cold 10%

glycerol (4°C)

Step.9 Dispense into 50 µl portions.

・electroporation

Step.1 Put 20 μl of 10% glycerol and 1 μl of the plasmid you want to put in a 0.6 ml tube.

Step.2 Pipetting

Step.3 Put the whole amount (50 μl) of the dissolved competent cell in a tube on ice.

Step.4 Tapping

Step.5 Place the entire amount in the cuvette for electroporation.

Step.6 Measure the impedance. Decrease the voltage (base is set to 1600v) by 200 until the inpedance is 1.

Step.7 Electroporation

Step.8 Immediately put 1ml of SOC in the cuvette and suspend

Step.9 Recovery incubation at 30°C for 1h

Step.10 Spread on appropriate plates.

https://static.igem.wiki/teams/4955/wiki/protocols/electroporation.pdf

Glycerol stock preparation from colonies

20% glycerol was used, which was dispensed and stored in 250 μl portions in a refrigerator at 4°C.

Loops were used to take the bacteria from the colonies, suspended in glycerol stock,

vortexed, and stored in a -80°C freezer.

Glycerol stock preparation from LB culture medium

80% glycerol was used, which was dispensed and stored in 250 µl portions in a refrigerator at 4°C.

After incubation with LB medium containing the appropriate bacteria overnight, 500 µl were suspended in 80% glycerol and vortexed strongly. It was then stored at -80°C.

https://static.igem.wiki/teams/4955/wiki/protocols/grist-preparation-from-lb-culture-medium.pdf

Extraction of Crocetin & Crocin

Step.1 Centrifuge 500 μl of the_ E. coli_ culture medium (6000g, 5 minutes) and discard the

supernatant.

Step.2 After suspending the cell pellets in 2ml of methanol (MeOH), extract them with an

ultrasonic disruption machine.

Step.3 Add Tris-HCl (50mM, pH 7.5) and vortex.

Step 4 Add 1M NaCl and vortex to confirm that it is dissolved.

Step.5 Centrifugal separation (15000rpm, 5min)

Step.6 Add 2ml of chloroform and vortex for 5 minutes

Step.7 Centrifugation (room temperature, 15,000 rpm, 15 min)

Step.8 Collect 1500 μl of the chloroform layer.

Step.9 Dry by centrifugal evaporation.

https://static.igem.wiki/teams/4955/wiki/protocols/extraction-of-crocetin-crocin.pdf

HP-LC-MS/MS analysis

Liquid chromatography was performed on a SHIMADZU Ultra-Fast Liquid　Chromatography

(UFLC) Nexera system (Shimadzu, Kyoto). Mobile phase conditions were A 0.1% formic

acid H2O, B 0.1% formic acid acetonitrile (10-100% B over 5 min, 100% B for 2.5 min, and

then 10% B for 2.5 min).

The mass spectrometer was a SCIEX Triple TOF X500R system (Sciex, Tokyo).

https://static.igem.wiki/teams/4955/wiki/protocols/hp-lc-ms-ms-analysis.pdf

Culture for fluorescence microscopy

5 mL of LB liquid medium was placed in a test tube and the appropriate antibiotic was added in the appropriate amount.

Amount of antibiotic added

2.5 μl of Carbenicillin 100μg/ml

6.25 μl of Tetracycline 10μg/ml

5 μl of Spectinomycin 100μg/ml

5 μl of Gentamicin 7μg/ml

The bacteria were suspended in liquid medium and incubated at 37°C for 18-24 hours with

horizontal shaking as a pre-culture.

In the main culture, 1 ml of pre-culture, 49 ml of TB liquid medium and 50 ml of final were

placed in a 300 ml flask and shaken horizontally at 150 rpm at 25°C until the OD value

reached 8 to 12.

During the main incubation, 0.1mM IPTG was added when the OD value reached 8, and

1μM Crystal Violet was added to induce nucleation.

Every 12 hours, 200 μl were sampled from 72~96 hours and stored at -20°C.

https://static.igem.wiki/teams/4955/wiki/protocols/culture-for-fluorescence-microscopy.pdf

Fluorescence Microscopy

Sample Preparation

OD measurement was performed prior to preparation of plates for observation.

The target Culture was sampled at 500 µl and vortexed with 2.5 µl of DAPI kept in a

refrigerator at 4°C for 30 s. The DAPI was added on ice.

The light was shaded and allowed to stand at room temperature for 5 min.

Centrifuged at 5000 rpm for 3 min at room temperature and discarded 300 µl of supernatant.

Ten μl of POLY-L-LYSINE stored in a refrigerator at 4°C was sprinkled on ice onto a preparat and spread over the entire surface using a tip.

The spread surface was placed face down and stood in a tip case or other container to allow the POLY-L-LYSINE to dry.

Once the POLY-L-LYSINE was completely dry, 5 μl of the sample adjusted as described

above was spread and a cover glass was placed.

The cover glass was pressed against the cover glass using a Kimwipe, and any overflowing liquid was wiped off with the Kimwipe.

The gap between the cover glass and the preparation was applied with clear nail polish to seal the gap and left at room temperature until completely dry.

How to use the Keyence fluorescence microscope

The microscope and PC were started up, and BZ-X Viewer was launched.

Selected still image capture.

The objective lens was set to 100x oil immersion lens, the microscope cover was opened,

and a drop of oil was placed on the tip of the objective lens.

A preparation was set and the tip of the lens was aligned with the location to be observed.

When observing, bright field (CH4) was selected, multicolor was enabled, and overlay was

set to disabled.

Focus was set so that the fungus was clearly visible.

If the number of visible bacteria was low, the supernatant was discarded after centrifugation

at 5000 rpm for 5 min at room temperature in an appropriate volume.

If the amount of bacteria was too large to observe cleanly, it was suspended in TB.

Fluorescence was set to blue (CH3), and the fluorescence exposure time was set to CV+- of the sample at the same time.

Measure was clicked, the shape was set to bar, the scale width was set to 2, numerical

display was selected, and overlay was selected when photographing, and four images were taken.

The images were saved by date of shooting with a file name indicating “time of incubation,

type of sample, how many”.

After observation, the PC was shut down and the microscope was turned off on the unit.

https://static.igem.wiki/teams/4955/wiki/protocols/fluorescence-microscopy.pdf

How to use imageJ

https://static.igem.wiki/teams/4955/wiki/protocols/imagej.pdf

Enzymatic reaction in E. coli crushed samples

Preparation of Magic Media™ medium

3.9 g of Invitrogen™‘s MagicMedia™ component A (product number (catalog number): K6803)was suspended in 95 mL deionized water and autoclaved at 121°C for 15 minutes. After cooling to 37°C, 5 ml of component B was added using aseptic technique. Storage was done at room temperature.

Reference

https://www.thermofisher.com/order/catalog/product/jp/ja/K6803