RPS detailed method and result

1. LasR repression

1.1. 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)

1.2. 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.

1.3. Result

1.4. Discussion

Judging from the result that fluorescence intensity of 3O-C12-HSL+ was lower than that of 3O-C12-HSL- in Pλ-rbs-gfp on pAC, we found that LasR was working. Because the regulator part used in this assay has been constructed from the regulator part of Ptrc-rbs-lasR-TT on pBR used in our lasI promoter assay (Previous & New), this result means that the regulator part of Ptrc-rbs-lasR-TT on pBR used in our lasI promoter assay (Previous & New) is working.

2. Previous lasI promoter activity

2.1. 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)

2.2. 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.

2.3. Result

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. lux-lac hybrid 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

5. Previous lsrA promoter activity and our new lsrA promoter activity

5.1. Sample

• Ptet-gfp on pSB1A2(JD22597)(positive control)
• Promoterless-gfp on pSB6A1(JD22597)(negative control)
• PlsrA-gfp on pSB1A2 (BBa_K649104)(JD22597)
• PlsrA-gfp on pSB1A2 (BBa_K11702-gfp)(JD22597)

5.2. Method

1. Overnight cultures of reporter strains grown at 37 °C in LB medium containing appropriate antibiotics were diluted 1:100 into 3 ml of LB medium and were incubated at 37 °C as fresh cultures.
2. After their OD590 reached 0.15, the fresh cultures were diluted 1:100.
3. After 4-hour incubation at 37 °C, 1 ml of each culture was moved to 1.6ml tube and its fluorescence intensity was measured with a flow cytometer.

5.3. Result

5.4. AI-2 working system

First AI-2 is synthesized by LuxS and accumulates outside the cell. Then AI-2 is imported into the cell by the LsrACDB transporter. Inside the cell, AI-2 is phosphorylated by the LsrK kinase. Phospho-AI2 relieves LsrR inhibition from the lsr operon, therefore activating PlsrA.

5.5. Discussion

To confirm that lsrA promoter(BBa_K117002) does not work properly, we introduced a gfp gene(BBa_J54103) downstream of the promoter and transformed this construct into JD22597 lacking lsrR. As a consequence, fluorescence intensity of lsrA promoter-gfp((BBa_K117002)-gfp) was almost the same as promoterless-gfp(negative control), showing that lsrA promoter (BBa_K117002) does not work properly. On the other hand, fluorescence intensity of our lsrA promoter-gfp(BBa_649104) was much higher than that of promoterless-gfp(negative control), showing that our new lsrA promoter (BBa_649100) works. The difference between lsrA promoter(BBa_K117002) and lsrA promoter (BBa_649100) is whether promoter contains CRP binding site(Fig) or not. Our lsrA promoter(BBa_649100) contains this site. According to Wang (2004) et al(ref.3), cAMP-CRP directly binds to the upstream of promoter and stimulates expression of the lsr operon.

Fig.CRP recognition sites are shown in capital letter.BBa_K649100 contains this sites

5.6. References

1. Karina B.Xavier and Bonnie L.Bassler 2004. Regulation of Uptake and Processing of the Quorum-Sensing Autoinducer AI2 in Escherichia coli. JOURNAL OF BACTERIOLOGY, Jan. 2005, p. 238-248
2. Ting Xue, Liping Zhao, Haipeng Sun, Xianxuan Zhou, Baolon Sun 2009. LsrR-binding site recognition and regulatory characteristics in Escherichia coli AI-2 quorum sensing. Cell Research (2009), p.1258-1268
3. Liang Wang, Yoshifumi Hashimoto, Chen-Yu Tsao, James J.Valdes, and William E.Bentley 2004. Cyclic AMP(cAMP) and cAMP Receptor Protein Influence both Synthesis and Uptake of Extracellular Autoinducer 2 in Escherichia coli. JOURNAL OF BACTERIOLOGY, Mar. 2005, p. 2066-2076

6. LsrR repression

6.1. Sample

• Ptet-gfp on pSB6A1(JM2.300)(positive control)
• Promoterless-gfp on pSB6A1(JM2.300)(negative control)
• PlsrA-gfp on pSB3K3(MG1655)(BBa_649104)
• PlsrA-gfp-PlsrR-lsrR on pSB3K3(BBa_649105)(MG1655)

6.2. Method

1. Overnight cultures of reporter strains grown at 37 °C in LB medium containing appropriate antibiotics were diluted 1:100 into 3 ml of LB medium and were incubated at 37 °C as fresh cultures.
2. After their OD590 reached 0.15, the fresh cultures were diluted 1:100.
3. After 4-hour incubation at 37 °C, 1 ml of each culture was moved to 1.6ml tube and its fluorescence intensity was measured with a flow cytometer.

6.3. Construction

PlsrA(BBa_K649100) and PlsrR-lsrR-lsrK(BBa_K649101) were amplified by PCR using the MG1655 as a template. PlsrA was ligated with igem part(BBa_E0040). Then PlsrR-lsrR-lsrK(BBa_K649101) was ligated into PlsrA-gfp(BBa_649104)

6.4. Result

Fig.1 the intensity levels of GFP fluorescence(after 4hr of dilution, dilution rate 1:100)

Fig.2 the intensity levels of GFP fluorescence(after 4hr of dilution, dilution rate 1:10)

6.5. References

1. Karina B.Xavier and Bonnie L.Bassler 2004. Regulation of Uptake and Processing of the Quorum-Sensing Autoinducer AI2 in Escherichia coli. JOURNAL OF BACTERIOLOGY, Jan. 2005, p. 238-248
2. Ting Xue, Liping Zhao, Haipeng Sun, Xianxuan Zhou, Baolon Sun 2009. LsrR-binding site recognition and regulatory characteristics in Escherichia coli AI-2 quorum sensing. Cell Research (2009), p.1258-1268
3. Liang Wang, Yoshifumi Hashimoto, Chen-Yu Tsao, James J.Valdes, and William E.Bentley 2004. Cyclic AMP(cAMP) and cAMP Receptor Protein Influence both Synthesis and Uptake of Extracellular Autoinducer 2 in Escherichia coli. JOURNAL OF BACTERIOLOGY, Mar. 2005, p. 2066-2076