From 2011.igem.org

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.
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.
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).
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. Results

1.4. Discussion

The above construction proves that the lasR part is working properly, which leads to our conclusion that the PlasI of the BBa_J64010 part is a defective promoter. The regulator part has the constitutive promoter Ptrc, so the regulator LasR is constantly being produced. LasR needs of 3OC12-HSL to bind to Plas and activate it. Therefore, if the LasR part is working, it should activate the Plas promoter when 3OC12-HSL is added, and cI should be produced. The reporter part is has a λ promoter, which is a constitutive promoter. This means gfp is constantly being expressed and fluorescence is observed. Since cI represses Pλ, a working LasR part should lead to a dropdown of the levels of fluorescence. As can be seen below in the results from our experiments, the fluorescence levels dropped down when we added 3OC12-HSL, which leads to the conclusion that the LasR part we used works properly.

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

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.
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.
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).
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. Results

2.4. Discussion

Because the regulator part used in our lasI promoter assay has been constructed from the regulator part used in our LasR repression assay, the regulator part used in lasI promoter assay is working. Nevertheless, fluorescence intensity of PlasI(BBa_J64010)-rbs-gfp-TT was not changed before and after 3O-C12-HSL induction. 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

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.
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.
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).
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. Results

4. lux-lac hybrid promoter activity

4.0. The AND-gate Mechanism

We designed AND-gate promoters that use two operators: one that uses activators as regulators, and other that uses repressors as regulators. After an activator binds to an inducer the resulting complex binds to the corresponding operator and activates it. However, since the AND-gate promoter needs its both operators to be active, transcription will not start until the operator that uses repressors as regulators has been de-repressed by the appropriate signaling molecule. This mechanism assures that transcription will not start until both players have added the corresponding signaling molecules to the place where the Judge bacterium is, so it fits perfectly in our RPS game design. To see the game results easily, we added a reporter gene downstream of the AND-gate promoter, so either of gfp, rfp or cfp is expressed when humans win, lose or tie the game, respectively.）

4.1. Sample

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

4.2. Method

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..
After their OD₆₀₀ reached 0.2, we added inducers into the fresh culture: 1 mM IPTG and/or 10 nM 3OC6-HSL. .
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).
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. Results

4.4. Discussion

4.5. references

Shotaro Ayukawa et al. "Construction of a genetic AND gate under a new standard for assembly of genetic parts" BMC Genomics (2010)
Robert Sidney Cox, III et al. "Programming gene expression with combinatorial promoters" molecular system biology (2007)
Rolf Lutz et al. "Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements" Nucleic Acids Research (1997)

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

5.0. 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.

5.1. 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

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.
After their OD590 reached 0.15, the fresh cultures were diluted 1:100.
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. Results

Fig. Not working lsrA promoter(BBa_K117002) and our new lsrA promoter(BBa_K649100)

5.4. 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

5.5. References

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
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
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

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.
After their OD590 reached 0.15, the fresh cultures were diluted 1:100.
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) ="construction"

6.4. Results

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

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
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
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

Team:Tokyo Tech/Projects/RPS-game/assay