Team:Gunma/Notebook

LABNOTES

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LABNOTES in Proof Of Concept

1. Crystal violet assay

Day 1

1.　The E. coli RP437 strain was statically grown at 37 °C overnight in Luria-Bertani (LB) medium*.

*Luria-Bertani (LB) medium (per 1 L):

• Yeast extract : 5 g
• Tryptone : 10 g
• NaCl : 10 g

Day 2

2. The overnight culture (2 μL) was diluted into each Onsen medium* (200 μL) in 96 well plates, then statically grown at 37 °C for 24 hours.

*Onsen medium (per 1 L):

• Original onsen water 950 mL
• 20% Glucose 25 mL
• 20% Casamino acid 25 mL

(For Watanoyu and Bandai media, pH was adjusted to 7.0 because they are highly acidic (pH < 2.5). Then, filtered.

Day 3

The optical density at 595 nm (OD595) was measured to monitor the cell growth.

Biofilm cells were washed five times with water to remove floating cells.

Biofilm cells were stained with 0.1% crystal violet for 10 minutes, and washed five times with water.

The crystal violet was eluted from biofilm cells by adding 95% ethanol.

The absorbance at 595 nm (A595) for each well was measured.

The capability for biofilm formation was determined as the A595 normalized to an OD595 of 1.

2. Biofilm 3D imaging

Day 1

1. The E.coli RP437 strain was statically grown at 37 °C overnight in LB medium.

Day 2

2. The RP437 overnight culture (30 μL) was diluted into a medium (3 mL), and inoculated on glass coverslips in a 6 well plate.

3. We grew the bacteria at 37 °C for 24 hours, statically.

Day 3

4. The biofilm cells on the coverslips were washed with phosphate buffered saline (PBS)* to remove the floating cells, and stained with 5 μM SYTO-9 (Life Technologies, Carlsbad, CA, USA) for 10 minutes.

5. The coverslips were washed twice, and fixed with 4% paraformaldehyde in PBS.

6. Fluoromount / Plus (Diagnostic BioSystems, Pleasanton, CA, USA) was mounted on a glass slide.

7. Fluorescent images were acquired in the Alexa Fluor 488 laser unit on an Olympus FV1200 IX81 microscope using a 60 X objective and captured with a charge-coupled-device (CCD) camera.

*Phosphate buffered saline (per 1 L):

• NaCl : 8 g
• Na₂HPO₄・12H₂O : 2.9 g
• KCl : 0.2 g
• KH₂PO₄ : 0.2 g
• Adjusted to pH 7.4

3. Preparation of bacterial cell lysate

1. Bacteria (DH5 α/pSB1C3 and DH5/α pSB1C3/α-gal-RFP) were aerobically pre-cultured overnight at 37 °C in 3 mL of LB medium.
2. The bacterial culture was inoculated into a fresh LB containing 0.2 mM IPTG, then aerobically grown at 25 °C for 24 hours in 5 mL of LB.
3. Bacterial cells were harvested and re-suspended in 800 μL of 50 mM Tris-HCl (pH 7.5).
4. The suspension was sonicated by ultrasound, divided into three 20 second intervals, and centrifuged at 20,000 g for 5 minutes at 4 °C.
5. The resulting supernatant was collected into a clean tube.
6. Proteins in the supernatant were quantified in a Bio-Rad protein assay according to the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA).

4. α-Galactosidase assay

1. The reaction was set up in a tube as follows:

• The recombinant α-galactosidase (6.78 mg/mL)　 2 μL
• 100 mM Dithiothreitol (DTT) 50 μL
• 100 mM MnCl₂ 50 μL
• 4 mg/mL *Oxidized NAD⁺ 25 μL
• 50 mM Tris-HCl (pH 7.5) 873 μL
• 4 mg/mL *PNPG substrate 200 μL

(* Oxidized nicotinamide adenine dinucleotide [NAD⁺])
(* p-Nitrophenyl α-D-Galactopyranoside [PNPG])

The reaction was incubated at 37 °C, 42 °C, 50 °C and 65 °C.
The enzyme reactions were monitored at 2 minutes intervals by measuring the absorbance at 420 nm.

5. Inhibition test of biofilm formation using the recombinant enzyme

Day 1

1. The E.coli RP437 strain was statically grown at 37 °C overnight in LB medium.

For Crystal violet assay:

Day2

2. The overnight culture (2 μL) was diluted into a fresh Ho-shi onsen medium (200 μL) , and statically grown at 37 °C for 24 hours.

Day3

3. The biofilm cells in a well were washed with phosphate buffered saline (PBS) to remove the floating cells. Hoshi onsen water containing 5 mM DTT, 5 mM MnCl, and 100 μg/mL oxidized NAD (NAD⁺) with and without α-Galactosidase (67.8 μg to 678 μg) was added into the biofilm cells, then we incubated it at 37 °C for 24 hours.

Phosphate buffered saline (per 1 L):

• NaCl : 8 g
• Na2HPO4・12H2O : 2.9 g
• KCl : 0.2 g
• KH₂PO₄ : 0.2 g
• Adjusted to pH 7.4

Day 4

4. Biofilm mass in bacterial cultures was measured by the crystal violet assay (as described in section 1)

For Biofilm 3D imaging:

Day2

2. The overnight culture (2 μL) was diluted into a fresh Ho-shi onsen medium (200 μL) , and statically grown at 37 °C for 24 hours.

Day3

3. The biofilm cells in a well were washed with PBS to remove the floating cells.

4. Hoshi onsen water containing 5 mM DTT, 5mM MnCl, and 100 μg/mL oxidized NAD (NAD⁺) with and without α-Galactosidase (67.8 μg to 678 μg) was added into the biofilm cells, then we incubated it at 37 °C for 24 hours.

Day 4

5. 3D images of the biofilm were acquired as described in section 2.

7. Degradation of biofilm using the bioengineered “suicide biofilm breaker” that overproduces α-galactosidase and along with SacB.

(a) Preparation for the biofilm breaker strain

Day 1

1. SacB / RFP-fused α-galactosidase-expressing SM10λ (carrying pABB-CRS2/sacB and pSB1C3/α-gal-RFP) and its control strain (SacB / RFP-expressing SM10λ: carrying pABB-CRS2/sacB and pSB1C3/RFP) were aerobically grown at 37 °C overnight in LB medium.

Day 2

2. The cultures were inoculated into 5mL LB media, and aerobically cultured in the presence of 0.2 mM IPTG (to induce α-galactosidase production) at 25 °C for 24 hours.

Day 3

3. Bacterial cells were harvested and re-suspended in 800 μL of 50 mM Tris-HCl (pH 7.5).

4. The resuspensions were incubated in Hoshi onsen water containing 5 mM DTT, 5 mM MnCl₂, and 100 mg/mL oxidized NAD (NAD⁺) and 30% sucrose (to induce cell lysis) at 25 °C for 2 hours.

(b) Biofilm assay

Day 1

5. The E. coli RP437 strain was statically cultured at 37 °C overnight in LB medium.

Day 2

6. The overnight culture was diluted into a fresh Ho-shi onsen medium, and statically grown at 37 °C for 24 hours.

Day 3

7. The biofilm cells were washed with PBS to remove the floating cells.

8. The biofilm breaker and its control strain were added, and incubated for another 24 hours in Hoshi onsen water containing 5 mM DTT, 5 mM MnCl₂, and 100 mg/mL oxidized NAD (NAD⁺) and 30% sucrose.

Day 4

9. Degradation of biofilm was estimated by crystal violet and 3D imaging assays (as described in section 1 and 2).

LABNOTES in Engineering Success

Expression of α-galactosidase-RFP (from E.coli)

We cloned the gene of α-galactosidase-RFP from E.coli into the pSB1C3 plasmid. This expression plasmid was transformed using DH5 α, and the transformant was cultured in liquid medium containing chloramphenicol. After culturing the transformant at 37 °C (130 rpm) for 16 hours, the cells were inoculated into a new medium and liquid-cultured until the logarithmic growth phase. IPTG was added to a final concentration of 0.2 mM, and the E.coli was cultured at 25 °C (130 rpm) for 20 hours. Cultured cells were sonicated with sonication buffer (phosphate buffer (pH 7.0) + 150 mM NaCl + 10% glycerol) and SDS-PAGE was performed.

The expression of α-Gal-RFP was verified by SDS-PAGE (Figure 1). A competent cell culture medium was used as a control or the wild type (WT).
Our target protein is about 76 kDa in molecular mass. In Figure 2, we observed that there was a thicker band in the vicinity of a band at 72 kDa than that of WT. Thus, we consider that this band represents α-Gal-RFP.

Figure 1. SDS-PAGE gel for quantification of α-galactosidase-RFP. M, molecular mass markers; WT, wild type; α-Gal-RFP, α-galactosidase-RFP from E.coli and coral; S, supernatant; P, pellet.

Expression of α-galactosidase (from Thermus thermophilus)

We cloned the gene of α-galactosidase from Thermus thermophilus into the pET28a plasmid. This expression plasmid was transformed using BL21(DE3), and the transformant was cultured in liquid medium containing kanamycin. After culturing the transformant at 37 °C (130 rpm) for 16 hours, the cells were inoculated into a new medium and liquid-cultured until the logarithmic growth phase. IPTG was added to a final concentration of 0.2 mM, and the E.coli was cultured at 25 °C (130 rpm) for 20 hours. Cultured cells were sonicated with sonication buffer (phosphate buffer (pH 7.0) + 150 mM NaCl + 10% glycerol) and SDS-PAGE was performed.

The expression of α-Gal was verified by SDS-PAGE (Figure 2). A competent cell culture medium was used as a control or the wild type (WT).
Our target protein is about 49 kDa in molecular mass. In Figure 2, we observed that there was a thicker band in the vicinity of a band at 52 kDa than that of WT. Thus, we consider that this band represents α-Gal.

Figure 2. SDS-PAGE gel for quantification of α-galactosidase. M, molecular mass markers; WT, wild type; α-Gal (T.t), α-galactosidase from Thermus thermophilus; P, pellet; S, supernatant.

Protein Dialysis_ α-Gal-RFP

To dialyze α-Gal-RFP, the protein was put in a dialysis tube (MWCO.50,000, SPECTRUM). The dialysis tube was immersed in dialysis buffer and stirred at 4 °C. The buffer was changed, as below. The composition of the dialysis buffer is as follows.

Dialysis buffer:

(1)

• 25 mM HEPES-KOH (pH 7.6)
• 500 mM KCl
• 2 mM EDTA
• 1 mM DTT
• 10% (w/v) glycerine

(2)

• 25 mM HEPES-KOH (pH 7.6)
• 500 mM KCl
• 2 mM EDTA
• 150 mM NaCl
• 5 mM MgCl₂・6H₂O

Exchanging dialysis buffer:

Protein concentration:

67.8 mg/mL

Figure 3. SDS-PAGE gel for quantification of dialyzed α-galactosidase-RFP. M, molecular mass markers; α-Gal-RFP, α-galactosidase-RFP from E.coli and coral after dialyzing; S, supernatant.

Protein Dialysis_ α-Gal from T.t

To dialyze α-Gal from Thermus thermophilus, the protein was put in a dialysis tube (MWCO.10,000, SPECTRUM). The dialysis tube was immersed in dialysis buffer and stirred at 4 °C. The buffer was changed, as below. The composition of the dialysis buffer is as follows.

Dialysis buffer:

(1)

• 25 mM HEPES-KOH (pH 7.6)
• 500 mM KCl
• 2 mM EDTA
• 1 mM DTT
• 10% (w/v) glycerine

(2)

• 25 mM HEPES-KOH (pH 7.6)
• 500 mM KCl
• 2 mM EDTA
• 150 mM NaCl
• 5 mM MgCl₂・6H₂O

Exchanging dialysis buffer:

Protein concentration:

65.36 mg/mL

Figure 4.SDS-PAGE gel for quantification of dialyzed α-galactosidase. M, molecular mass markers; α-Gal (T.t), α-galactosidase from Thermus thermophilus after dialyzing; S, supernatant.

Colonies of cells harbouring the plasmid expressing α-galactosidase-RFP (from E.coli) on agar plates

E.coli DH5 α strain was transformed with pSB1C3/ α-galactosidase-RFP onto LB agar plates containing chloramphenicol, which was incubated for 20h at 37 °C until becoming colonies. Moreover, we continued incubating the plates for 24 h at 37 °C until colonies were red.

In Figure 5, we observed both red and white colonies. This fused protein contains RFP, We found that red colonies are usually smaller than white ones.

Figure 5. Expression of α-galactosidase-RFP in E.coli DH5 α strain.