Experiments

The plasmids related to this project were constructed using Snap Gene, a software for everyday molecular biology.

The plasmid parts we designed were manufactured by Twist bioscience and Integrated DNA technology(IDT).

PCR for cloning: We amplified the parts (backbone, fragment) required for plasmid construction using PCR. Primers varied depending on what we amped.

Prepare Premix as above in PCR tube

1.Add MQ to 25μl

2.Add optional DNA

3.The DNA was amplified using the following thermal cycling conditions:

[ ・98℃ 30 sec

・98℃ 10 sec

・66℃ 20 sec

・72℃ vary depending on template DNA ] 35 cycle

72℃ 2 min

4℃ ∞

Gels were created for electrophoresis as follows:

1.Dissolve agarose S in 1x TAE buffer at a rate of 1% and heat the mixture until agarose dissolves.

2.Pour the mixture into a conical tube.

3.Add EtBr at a 1:10000 ratio to the gel, and gently mix and tumble ten times to ensure even distribution.

4.Pour the gel into a tray and make it solidify at room temperature.

※ 1x TAE buffer is prepared by diluting 50x TAE buffer which has 2M tris-acetate and 0.05M EDTA. 10 mg/mL EtBr was purchased from Fujifilm.

This procedure was conducted to assess the length of DNA fragments which are amplified through techniques such as PCR.

1.Place the gel in the electrophoresis chamber without removing it from the tray.
2.Pour 1x TAE buffer into the chamber until the gel is fully immersed.
3.Add an appropriate size marker to the first lane.
4.Apply the samples (the quantity depends on each reagent) sequentially in the lanes, starting from the second lane.
5.Turn on the chamber and run electrophoresis at 100 V for 20 min.
6.Remove the gel carefully from the tray.
7.Visualize the bands using a trans illuminator.

Use two types of markers depending on the target DNA strand length, and dye the migrated DNA with 6x loading dye.

Isolate the desired fragments of undamaged DNA from the agarose gel after gel electrophoresis.

Gibson Assembly …

This is a technique for assembling DNA seamlessly and in the correct order based on exonucleases.

1.Set up the following reaction on ice:

2.Incubate samples in a thermal cycler at 50℃for 15 minutes when 2 or 3 fragments are being assembled.

Gibson Assembly Master Mix (Q5 High-Fidelity 2X Master Mix) contains dNTPs, Mg++, and a dedicated buffer. Simply add primers and DNA templates to enable robust amplification regardless of GC content

Goldengate Assembly …

Goldengate Assembly can be used for directed assembly of multiple inserts/modules and single insert/library generation cloning with single insert(s) using the Golden Gate approach.

1.Set up assembly reactions as follows:

2.For 2-10 inserts, (37°C, 1 min → 16°C, 1 min) x 30 → 60°C, 5 min

We introduced the plasmid into E. coli following this protocol. We used two strains of E. coli: DH5α (the strain commonly used for routine transformation) and NEB5α (the strain recommended by NEB).

DH5α

1. Mix 2μl of plasmid and 50μl of E. coli + pipetting or flicking

2. Leave the mixture on ice for 10 min

3. Place the mixture in a heat block at 42℃ for 45 sec

4. Leave it on ice for 2 min

5. Drip 50μl of the mixture on an optional plate

6. Incubate the plate at 37℃ for 11h~15h

7. After checking the colony grows, store at 4℃

If recovery culture was required (in a case we use Cm as an antibacterial substance), after step 4, we added 950μl of SOC into the mixture. After that, we shake it vigorously at 37℃.

NEB5α

1. Thaw a competent E. coli

2. Add 2μl of the chilled DNA to the cell mixture

3. Pipetting or flicking 4-5 times to mix cells and DNA

4. Place it on ice for 30 min

5. Place the mixture in a heat block at 42℃ for exactly 30 sec

6. Leave it on ice for 2 min

7. Mix the cells thoroughly by flicking the tube and inverting

8. Drip 50 ~100μl of each dilution on a selection plate

9. Incubate the plate at 37℃ for 11h~15h

10. After checking the colony grows, store at 4℃

If recovery culture is required (in a case we use Cm as an antibacterial substance), after step 6, we added 950μl of SOC into the mixture. After that, we shake it vigorously at 37℃.

We used LB Broth, Miller (The composition of the LB Broth MILLER per liter is 5g yeast extract, 10g peptone from casein, and 10g sodium chloride.)

1.A mixture is prepared as follows:

2.Heat in the microwave until transparent.

3.Once cooled, add optional antibacterial agents and pour onto a plate to solidify.

1. A single, relatively isolated colony was picked using a pipette tip, and the tip was dipped into the liquid which is prepared as follows:

2. Dispense 100µL of LB medium (no antibiotics) to 1.5 mL tube. Incubate at 37℃ for 1 h

Prepare premix and dispense 9.75 µl each into PCR tubes

Add the template (culture) 1 µL

The DNA was amplified using the following thermal cycling conditions:

[・98℃ 30 sec

・98℃ 10 sec

・66℃ 20 sec

・72℃ 1min 52sec ] 35 cycle

72℃ 2 min

4℃ ∞

1.Add 3 ml of LB and the optional antimicrobial agent to a 15 ml tube.

2. Take a small amount of glycerol stock and suspend it.

3.Incubate at 37℃ O/N (12h～)

We take this process to extract the plasmid, using Fast Gene Plasmid mini kit.

1.Harvest of bacteria: c.f.g. 10,000 rpm; 1 min → remove the sup

2.Lysis: add 200μL of mp1 → voltexing, add 200μL of mp2: invert the tube → stay 2min at RM, add 300μL of mp3: invert the tube

3.Lysate: clarification: c.f.g.13,000 rpm; 2 min

4.Sample loading: Load the sup to the column → c.f.g 13,000 rpm; 2 min

5.Membrane washing and membrane drying: add 150μL of mp4 and 300μL of mp5 → 13,000 rpm; 3 min

6.Elation: 50μL of mp6 → stay 2 min at RM→ 13,000 rpm; 2 min

We conducted sequence analysis using sequencing provided by Azenta Life Science and sequencing provided by Gifu University Institute for Advanced Studies Center for Scientific Research Infrastructure. All functional regions of the plasmid used in this project were subjected to sequence analysis to verify their sequences.

1.Add 3 ml of LB, 60μl of 25% glucose and the optional antimicrobial agent to a 15 ml tube.

2.Pick a single isolated colony and dip it into the liquid.

3.Shake culture 145rpm, 37℃, O/N

1. Dispense 20ml of LB and add Amp 20μl and glucose 400μL

2.Shake it at 37℃ 3h

We conducted this experiment with the assistance of Professor Takashi Yokogawa.

1. Prepare the separation gel solution and concentrated gel as follows:

2.add 40µl of 40% APS, 5µl of TEMED → pour into the gel plate and layer EtOH

3.After the separating gel solidifies → add 20µl of 40% APS, 2.5µl of TEMED to concentrated gel

4.pour into the gel plate → insert a comb

5.Apply marker and optional samples

1.Dilute each optional plasmid 5-fold with MiliQ

2.Mix plasmids

3.Mix plasmids and E. coli as follows:

→ pipetting or flicking

4.on ice for 5 min

5.heat shock 42℃ for 30 sec

6.Add 80μl of room temperature SOC into the mixture.

7.Place at 37℃ for 60 min → shake vigorously (250rpm) or rotate

8.plate on LB plate

9.Incubate at 37℃ for 11h～15h

1.37℃ 10min incubate

2.70℃ 10 min heat

Reagents:

・Cell extract (supernatant suspended in binding/wash buffer)

・Binding/Wash buffer: 50 mM potassium phosphate (NaPi) buffer [pH 7.4], 300 mM

NaCl, 20 mM imidazole

・Elution buffer: 250 mM and 500 mM imidazole

・Ni-NTA agarose

Procedure:

↓mAdded 100 mL of RO water to a tube containing 100 mL of resin.

↓Resuspended the resin and aliquoted.

↓Added 500 µL of cell extract (IPTG-induced and non-induced).

↓Kept on ice.

↓Gently inverted every 2-3 min for 10 min.

↓Transferred the entire mixture to a spin column.

↓Centrifuged using a benchtop centrifuge for 2-3 sec.

↓Collected the flow-through (Fl).

↓Added 0.6 mL of 20 mM His buffer onto the resin in the spin column.

↓Mixed gently by slow pipetting.

↓Centrifuged for 2-3 sec using a benchtop centrifuge.

↓Collected the wash fraction (Wa1).

↓Repeated steps 9-12 to obtain the second wash fraction (Wa2).

↓Added 200 µL of 250 mM imidazole, kept on ice for 2 min, and pipetted several times.

↓Centrifuged for 2-3 sec and collected the eluate (El1).

↓Added 500 mM imidazole (two additions of 200 µL 250 mM imidazole), kept on ice for 2 min and pipetted several times.

↓Centrifuged for 2-3 sec and collected the eluate (El2).

↓Confirmed the collected samples by SDS-PAGE.