RPS detailed method and result

1. Previous lasI promoter activity

1.1. Sample

• PlasI(BBa_J64010)-rbs-gfp-TT on pSB3K3 / Ptrc-rbs-lasR-TT on pBR
• promoterless-gfp on pSB3K3 / Ptrc-rbs-lasR-TT-PlasI-rbs-cI-TT on pBR (negative control)

1.2. Method

1. Overnight culture of sample strain grown at 37 °C in LB medium containing carbenicillin and kanamycin, and overnight culture of promoterless negative control grown at 37 °C in LB medium containing carbenicillin and kanamycin were diluted 1:300 in the medium, and then they were incubated at 37 °C as fresh cultures.
2. After their OD₆₀₀ reached 0.2, we added 3 µL of 500 µM 3O-C12-HSL (3OC12-HSL+) or 3 µL of DMSO (3OC12-HSL-) into the fresh cultures.
3. After 3-hour incubation at 37 °C (OD approximately reached 1.50.), 0.25 mL of each culture was harvested by centrifugalization and suspended by adding 1 mL of PBS (phosphate-buffered saline).
4. We dispensed 500 µL of each suspension into a disposable tube through a cell strainer, and its fluorescence intensity was measured with a flow cytometer of Becton, Dickinson and Company.

1.3. Result

2. LasR repression

2.1. Sample

• Ptrc-rbs-lasR-TT-PlasI-rbs-cI on pBR / Pλ-rbs-gfp on pAC
• Ptrc-rbs-lasR-TT on pBR / promoterless-rbs-gfp-TT on pSB3K3 (negative control)

2.2. Method

1. Overnight culture of sample strain grown at 37 °C in LB medium containing carbenicillin and chloramphenicol were diluted 1:20 in the medium, and overnight culture of promoterless negatgive control grown at 37 °C in LB medium containing carbenicillin and kanamycin were diluted 1:300 in the medium and then they were incubated at 37 °C as fresh cultures.
2. After their OD₆₀₀ reached 0.2, we added 3 µL of 500 µM 3O-C12-HSL (3OC12-HSL+) or 3 µL of DMSO (3OC12-HSL-) into the fresh cultures.
3. After 3-hour incubation at 37 °C (OD approximately reached 1.50.), 0.25 mL of each culture was harvested by centrifugalization and suspended by adding 1 mL of PBS (phosphate-buffered saline).
4. We dispensed 500 µL of each suspension into a disposable tube through a cell strainer, and its fluorescence intensity was measured with a flow cytometer of Becton, Dickinson and Company.

2.3. Result

2.4. Discussion

Judging from the result that fluorescence intensity of 3O-C12-HSL+ was lower than that of 3O-C12-HSL- in Sample 1, we found that LasR which was used for Sample 2 was working. Because the regulator part of sample 2 has been constructed from the regulator part of sample 1, this result means that the regulator part of sample 1 is working. Nevertheless, fluorescence intensity of 3O-C12-HSL+ and 3O-C12-HSL- in Sample 1 was not different. From this result, we considered that las promoter (BBa_J64010) was not regulated by lasR and 3O-C12-HSL.

3. New lasI promoter activity

3.1. Sample

• Ptrc-rbs-lasR-TT on pBR / PlasI(BBa_I649000)-rbs-gfp-TT on pSB3K3
• Ptrc-rbs-lasR-TT on pBR / promoterless-rbs-gfp-TT on pSB3K3 (negative control)

3.2. Method

1. Overnight cultures of sample strain grown at 37 °C in LB medium containing carbenicillin and kanamycin were diluted 1:100 in the medium, and overnight cultures of promoterless negative control strain grown at 37 °C in LB medium containing carbenicillin and kanamycin were diluted 1:200 in the medium , and then they were incubated at 37 °C as fresh cultures.
2. After their OD₆₀₀ reached 0.2, we added 3 µL of 500 µM 3O-C12-HSL (3OC12-HSL+) or 3 µL of DMSO (3OC12-HSL-) into the fresh cultures.
3. After 3-hour incubation at 37 °C (OD reached approximately 1.80.), 0.25 mL of each culture was harvested by centrifugalization and suspended by adding 1 mL of PBS (phosphate-buffered saline).
4. We dispensed 500 µL of each suspension into a disposable tube through a cell strainer, and its fluorescence intensity was measured with a flow cytometer of Becton, Dickinson and Company.

3.3. Result

4. New lasI promoter activity

4.1. Sample

• I751101 on pSB3K3 / pTrc99A
• promoterless-rbs-gfp-TT on pSB3K3 / pTrc99A (negative control)

4.2. Method

1. Overnight culture of BBa_I751101 grown at 37 °C in LB medium containing carbenicillin and kanamycin, and overnight culture of promoterless negative control grown at 37 °C in LB medium containing carbenicillin and kanamycin were diluted 1:100 in the medium, and then they were incubated at 37 °C as fresh cultures..
2. After their OD₆₀₀ reached 0.2, we added inducers into the fresh culture: 1 mM IPTG and/or 10 nM 3OC6-HSL. .
3. After 3-hour incubation at 37 °C (OD approximately reached 1.70.), 0.25 mL of each culture was harvested by centrifugalization and suspended by adding 1 mL of PBS (phosphate-buffered saline).
4. We dispensed 500 µL of each suspension into a disposable tube through a cell strainer, and its fluorescence intensity was measured with a flow cytometer of Becton, Dickinson and Company.

4.3. Result