RPS detailed method and results

1. LasR repression

1.1 Plasmid transformed into E.coli

• Sample
  
  1. Ptrc-rbs-lasR-TT-PlasI-rbs-cI on pBR
     
  2. Pλ-rbs-gfp on pAC
     
• negative control
  
  1. Ptrc-rbs-lasR-TT on pBR
     
  2. promoterless-rbs-gfp-TT on pSB3K3

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 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 Plasmid transformed into E.coli

• Sample
  
  1. PlasI(BBa_J64010)-rbs-gfp-TT on pSB3K3
     
  2. Ptrc-rbs-lasR-TT on pBR
     
• negative control
1. promoterless-gfp on pSB3K3
2. Ptrc-rbs-lasR-TT-PlasI-rbs-cI-TT on pBR
   

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 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 Plasmid transformed into E.coli

• Sample
  
  1. Ptrc-rbs-lasR-TT on pBR
     
  2. PlasI(BBa_K649000)-rbs-gfp-TT on pSB3K3
     
• negative control
  1. Ptrc-rbs-lasR-TT on pBR
     
  2. promoterless-rbs-gfp-TT on pSB3K3

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 Results

3.4 References

1. Patrick C. Seed et al. “Activation of the Pseudomonas aeruginosa lasI Gene by LasR and the Pseudomonas Autoinducer PAI: an Autoinduction Regulatory Hierarchy” JOURNAL OF BACTERIOLOGY (1995)

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 Plasmid transformed into E.coli

• Sample
  
  1. I751101 on pSB3K3
     
  2. pTrc99A
• negative control
1. promoterless-rbs-gfp-TT on pSB3K3
2. pTrc99A

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 Results

4.4 Discussion

In the absence of the both inducers, the culture with Plux-lac hybrid promoter-gfp showed the background–fluorescence intensity generated by promoterless-rbs-gfp on pSB3K3. The presence of either IPTG or AHL alone had little effect on increasing the fluorescence intensity. In the presence of both inducers, the culture showed about 100-fold higher fluorescence intensity than that in the absence of both inducers. This result confirmed that the assembly of the two LacI operators and the LuxR operator integrated the inputs of IPTG and 3OC12-HSL into the output of GFP transcription. In other words, combination of the two LacI operators and the LuxR operator functioned as a genetic AND gate.

4.5 References

1. Shotaro Ayukawa et al. “Construction of a genetic AND gate under a new standard for assembly of genetic parts” BMC Genomics (2010)
2. Robert Sidney Cox, III et al. “Programming gene expression with combinatorial promoters” molecular system biology (2007)
3. 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 lsrA promoter.

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_K117002)-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 1.6 mL tube and its fluorescence intensity was measured with a flow cytometer.

5.3 Results

Fig 5.1 Not working lsrA promoter(BBa_K117002) activity and our new lsrA promoter activity(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. In spite of no LsrR repression, gene transcription does not take place sufficiently. On the other hand, fluorescence intensity of our lsrA promoter-gfp(BBa_K649104) was much higher than that of promoterless-gfp(negative control), showing that our new lsrA promoter (BBa_K649100) works. Gene transcription takes place sufficiently. The difference between lsrA promoter(BBa_K117002) and lsrA promoter (BBa_K649100) is whether promoter contains CRP binding site(Fig 5.2) or not. Our lsrA promoter(BBa_K649100) 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 5.2 CRP recognition sites are shown in capital letter. Our new promoter lsrA(BBa_K649100) contains this sites

5.5 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

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_K649104)
  
• PlsrA-gfp-PlsrR-lsrR on pSB3K3(BBa_K649105)(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 1:10.
3. After 4-hour incubation at 37°C, 1 mL of each culture was moved to 1.6 mL tube and its fluorescence intensity was measured with a flow cytometer.

6.3 Construction of PlsrA-gfp-PlsrR-lsrR

PlsrA(BBa_K649100) and PlsrR-lsrR(BBa_K649101) were amplified by PCR using the MG1655 as a template. PlsrA was ligated with igem part(BBa_E5500). Then PlsrR-lsrR(BBa_K649101) was ligated into PlsrA-RBS-gfp(BBa_K649104).
This part(BBa_K649105) is described as PlsrA-gfp-PlsrR-lsrR because lsrK of BBa_K649101 has mutation and does not work and other genes work.

6.4 Results

Fig 6.1 the intensity level of GFP fluorescence

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

7. in vitro assay for lox2272

7.1 Construction of lox2272-gfp-lox2272

PlacIQ-lox2272-gfp-lox2272(BBa_K649200) is composed of lox2272(BBa_K649204) and PlacIQ-gfp(BBa_J54202). PlacIQ-gfp contains Bgl II and Mlu I restriction site on upstream of gfp. These restriction sites allows insertion of the upstream lox2272, designed to be flanked by the sticky ends of BamH I and Mlu I. This lox2272 was inserted to the PlacIQ-gfp digested with Bgl II and Mlu I. Likewise, the down stream lox2272 was inserted between Spe l and Pst l by same means.

7.2 Sample

sample	volume
PlacIQ-lox2272-gfp-lox2272(pSB1C3)	50 ng/µL × 4 µL
10× Cre recombination Buffer	1 µL
ddH2O	4 µL
Cre recombinase	1,000 units/mL × 1 µL
total	10 µL

7.3 Method

1. The part on pSB1C3 was made linear by EcoRV restriction site which is far from lox sites.
2. Three identical samples and one negative control supplied with ddH₂O instead of Cre-recombinase had been prepared and those were incubated in 37°C for 0.5hr, 2hr and 4hr respectively. Negative control was incubated for 4hr.
3. After a period of time, the samples were kept in -20°C until the whole samples are arranged. The result was checked by electrophoresis(0.8% EtBr(+) agarose gell, 100 V, 400 mA, 30 min).

7.4 Results

From left, each lane corresponds to negative control, 0.5hr, 2hr and 4hr respectively. It was confirmed by earlier control experiment that several bands appear when recombination happens. As you can see in the picture, there are three bands shown in period of 2hr and 4hr. It can be said that excision at lox2272 sites did happen.

7.5 References

1. Shotaro Ayukawa, Akio Kobayashi, Yusaku Nakashima, Hidemasa Takagi, Shogo Hamada, Masahiko Uchiyama, Construction of a genetic AND gate under a new standard for assembly of genetic parts,BMC Genomics. 2010; 11
2. Kimi Araki, Masatake Araki1 and Ken-ichi Yamamura, Site-directed integration of the cre gene mediated by Cre recombinase using a combination of mutant lox sites, Nucleic Acids Research, 2002.

8. in vivo assay for lox cassettes

8.1 Sample

Two kinds of BioBricks were prepared for the assay.
One was PlacIQ-lox2272-rfp-lox2272-gfp(BBa_K649201),
and the other was PlacIQ-lox71-rfp-lox66-gfp(BBa_K649202)
The assay for these parts was carried out simultaneously.

8.2 Method

The method for two BioBricks' assay was same as below.

1. Strain JM2.300 with both P_BAD/araC-Cre and PlacIQ-lox-rfp-lox-gfp in it was cultured at 37°C in 3 mL of LB medium containing ampicillin (6 µL) and kanamycin(3.6 µL). Same kind of strain having only PlacIQ-lox-rfp-lox-gfp was cultured in same volume of LB containing kanamycin (3.6 µL) and carbenicillin(4 µL). These were cultured until OD 1.6.
2. Each cultured medium was 6 times diluted in the medium and three samples (3 mL each) were dispensed from those. Among them, two were induced by arabinose (2 M, 75 µL).
3. After 30 min from induction, 1000 times dilution and 100000 times dilution of each sample were plated and incubated in 37°C about 12 hours. The florescence of plates was examined by FLA and it was taken picture. About the rest of the samples, strains were harvested by centrifugation and suspended by adding 1 mL of PBS (phosphate-buffered saline). The last OD of PBS solution was approximately 0.4. We dispensed 700 µL of each suspension into a disposable tube through a cell strainer, and fluorescence intensity of each cell was measured with a flow cytometer of Becton, Dickinson and Company.
4. In the same way as 3, 9 more samples(for each BioBrick) were examined at period 1hr, 2hr, and 4hr.

8.3 Results

(a)

(b)

(c)

Fig 8.1 Cre-meditated recombination at lox2272 cassette. Cre-induction period of 0.5 hr.(a) Overlay of Green and Red channel. The leftmost is a negative control which don't have Cre-expressing plasmid. The center is an arabinose induced sample which has both Cre plasmid and BioBrick K649201. The rightmost is a uninduced strain which has both plasmid like as the center. (b)Detection of GFP. The order of samples is same as above.(c)Detection of mCherry. The order of samples is same as above.

(a)

(b)

(c)

Fig 8.2 Cre-meditated recombination at lox71/66 cassette. Cre-induction period of 0.5 hr.(a) Overlay of Green and Red channel. The leftmost is a negative control which don't have Cre-expressing plasmid. The center is an arabinose induced sample which has both Cre plasmid and BioBrick K649202. The rightmost is a uninduced strain which has both plasmid like as the center. (b)Detection of GFP. The order of samples is same as above.(c)Detection of mCherry. The order of samples is same as above.

(a)	(b)
(c)	Fig 8.4 identical plates with Fig 8.3 (a)expression levels of red and green florescence of K649201 (b)expression levels of red and green florescence of K649202 (c)examined area for comparing between red and green florescence at each plate

Fig 8.5 Green florescence level of each cell was detected by flow cytometer. (a)arabinose induced strain containig K649201 and cre-expressing plasmid (b)arabinose supplied strain containing only K649201 (c)arabinose induced strain containing K649202 and cre-expressing plasmid (d)arabinose supplied strain containing only K649202

8.4 Discussions

We made it clear at the previous page that two cassettes of different excision frequency are necessary for the adequate randomizer. We confirmed that our lox71/66 and lox2272 cassettes work in strain. In addition, we are informed that the recombination efficiency of lox71/66 is superior to that of lox2272 from the results of the experiments. It is clear that excision occurs more frequently as longer as the sequence flanked by lox cassette is. Therefore, it is reason to design the randomizer with these two cassettes as following.

lox71-lox2272-encoding gene-lox66-encoding gene-lox2272

By designing sequence flanked by lox2272 longer than that by lox71/66, we may operate the frequency equally.

8.5 References

1. Jean Livet, Tamily A. Weissman, Hyuno Kang, Ryan W. Draft, Ju Lu, Robyn A. Bennis, Joshua R. Sanes and Jeff W. Lichtman, Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system, Nature 2007.
2. Kimi Araki, Masatake Araki1 and Ken-ichi Yamamura, Site-directed integration of the cre gene mediated by Cre recombinase using a combination of mutant lox sites, Nucleic Acids Research, 2002.