Part:BBa_K1529302:Experience

Prhl(RL)-CmR-LasI

Materials and Methods

-Strain
All the samples were JM2.300 strain.

C4HSL-dependent CmR expression Protocol

1.Construction
A. Ptet-GFP-Ptet-RhlR (pSB6A1), Prhl(RL)-CmR-LasI(pSB3K3)
B. Ptet-GFP-Ptet-RhlR (pSB6A1), PlacIq-CmR (pSB3K3)…Positive control
C. Ptet-GFP-Ptet-RhlR (pSB6A1), promoter less CmR (pSB3K3)… Negative control

Fig. 1.Plasmids for the experiment of C4HSL-dependent CmR expression.

2.Assay protocol
1. Prepare overnight cultures for the sender cells in 3 mL LB medium, containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) at 37°C for 12h.
2. Make a 1:100 dilution in 3 mL of fresh LB containing antibiotic and grow the cells at 37°C until the observed OD590 reaches 0.5.(→fresh culture)
3. Add 30 microL of suspension in the following medium.
　　　1) 3 mL of LB containing Amp and Kan + 30 microL C4HSL (final concentration is 5 microM)
　　　2) 3 mL of LB containing Amp and Kan + 30 microL DMSO
　　　3) 3 mL of LB containing Amp, Kan and Cm (final concentration is 100 microg/mL) + 30 microL C4HSL (final concentration is 500 microM)
　　　4) 3 mL of LB containing Amp, Kan and Cm (final concentration of Cm is 100 microg/mL) + 30 microL DMSO
4. Grow the samples of sender cells at 37°C for more than 8 hours.
5. Measure optical density every hour. (If the optical density is over 1.0, dilute the cell medium to 1/10.)

C4HSL-dependent 3OC12HSL production Protocol

1.Construction
Sender
A. Ptet-GFP-Ptet-RhlR (pSB6A1), Prhl(RL)-CmR-LasI (pSB3K3)
D. Ptet-GFP-Ptet-RhlR (pSB6A1), Plux-CmR (pSB3K3)
Reporter
E. Ptrc-LasR (pSB6A1), Plas-GFP (pSB3K3)
F. Ptet-LuxR (pSB6A1), PlacIq-GFP (pSB3K3)...Positive control
G. Ptet-LuxR (pSB6A1), Promoter-less-GFP (pSB3K3)...Negative control

Fig. 2.Plasmids for the experiment of C4HSL-dependent 3OC12HSL production.

2.Assay protocol
Prepare the supernatant of the sender cell
1. Prepare overnight cultures for the sender cells in 3 mL LB medium, containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) at 37°C for 12h.
2. Make a 1:100 dilution in 3 mL of fresh LB containing antibiotic and grow the cells at 37°C until the observed OD590 reaches 0.5.
3. Add 30 microL of the culture containing the cells in the following medium.
　　　a) Add 15 microL of 10 mM C4HSL to 3 mL LB containing Amp and Kan (final concentration is 50 microM)
　　　b) Add 15 microL DMSO to 3 mL of LB containing Amp＋Kan
4. Grow the samples of sender cell at 37°C for 8 hours.
5. Measure the optical density every hour. (If the optical density is over 1.0, dilute the cell medium to 1/10.)
6. Centrifuge the sample at 9000x g, 4°C for 1 min. Filter sterilize the supernatant. (Pore size is 0.22 microm.)
7. Use the supernatant in reporter assay.

Reporter Assay
1. Prepare overnight cultures for the Reporter cell (E～G) in 3 mL LB medium, containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) at 37°C for 12h.
2. Make a 1:100 dilution in 3 mL of fresh LB + antibiotic and grow the cells at 37°C until you reach an 0.5 in OD590 (fresh culture).
3. Add 30 microL of suspension in the following medium.
　　　1) 2.7 mL filtrate of Aa +300 microL LB
　　　2) 2.7 mL filtrate of Ab +300 microL LB
　　　3) 2.7 mL filtrate of Da +300 microL LB
　　　4) 2.7 mL filtrate of Db +300 microL LB
　　　5) 3 mL LB + 5 microM C12HSL 3 microL (Final concentration is 5 nM)
　　　6) 3 mL LB + DMSO 3 microL
4. Grow the samples of Reporter cell in incubator at 37°C for 4 h.
5. Start preparing the flow cytometer 1 h before the end of incubation.
6. After the incubation, take the sample, and centrifuge at 9000x g, 1 min., 4°C.
7. Remove the supernatant by using P1000 pipette.
8. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend. (The ideal of OD is 0.3.)
9. Dispense all of each suspension into a disposable tube through a cell strainer.
10. Use flow cytometer to measure the fluorescence of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company.)

Results

C4HSL-dependent CmR expression Result

　We tested two types of culture condition which contains different concentration of chloramphenicol(Cm). (0 and 100 microg / mL)
Fig. 3, Fig. 4 shows the condition in the absence and presence of chloramphenicol, respectively.

• 

  Fig. 3. C4HSL-dependent CmR expressionh in no chloramphenicol

• 

  Fig. 4. C4HSL-dependent CmR expressionh in 100 microg / mL chloramphenicol

　Fig. 3 shows that every cell can grow in the absence of chloramphenicol.
　On the other hand, in the presence of chloramphenicol, the cell containing Prhl(RL)-CmR-LasI can grow only when induced by C4HSL.
Without the induction of C4HSL, the cell cannot express CmR and cannot grow in the presence of chloramphenicol.
As a result, we confirmed that Prhl(RL)-CmR-LasI expressed CmR when induced by C4HSL as expected.

C4HSL-dependent 3OC12HSL production Result

　Fig. 5 shows the fluorescence intensities generated by the reporter cells.
When the reporter cell E was incubated in the condition (1) (the culture of the induced Company cell), the fluorescence intensity of the reporter cell increased.
Comparing the results of condition (1) and (2) reporter cell in the supernatant of (1) had 29-fold higher fluorescence intensity.

　This result indicates that Company cell produced 3OC12HSL in response to C4HSL induction by the function of Prhl(RL)-CmR-LasI.
From this experiment, we confirmed that a new part Prhl(RL)-CmR-LasI synthesized 3OC12HSL (LasI) as expected.

Fig. 5. 3OC12HSL production in the presence of C4HSL

Co-culture Assay

　From the results so far, Company cell containing Prhl(RL)-CmR-LasI expresses CmR and LasI in the presence of C4HSL.
This enables Company to survive and to produce 3OC12HSL. (This result is showed above.)
On the other hand, Customer cell containing Plux-CmR-RhlI(BBa_K1529797) expresses CmR and RhlI in the presence of 3OC12HSL.
This enables Customer to survive and to produce C4HSL. (This result is showed here.)
These functions will make the mutualism of Company and Customer possible.

　To characterize the mutualism of Company cell and Customer cell, we cultivated both of them in different ratio and measured the growth.
We constructed the Company cell containing GFP and the Customer cell containing RFP.
After cultivating both of them for six hours in the same medium, we detected the mutualism by checking the fluorescence intensity of GFP with flow cytometer.
We measured the optical density every hour during the culturing.

Co-culture Assay Protocol

-Strain
All the samples were JM2.300 strain

1.Construction
A. Ptet-GFP-Ptet-RhlR (pSB6A1), Prhl(RL)-CmR-LasI (pSB3K3)
B. Ptet-LuxR-Plac-RFP (pSB6A1), Plux-CmR-RhlI (pSB3K3)

Fig. 6.Plasmids for the experiment of Co-culture.

2.Assay Protocol
1. Prepare overnight cultures for each samples A and B in 3 mL LB medium, containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) at 37°C for 12 h.
2. Dilute the overnight cultures to 1 / 100 in fresh LB medium (3 mL) containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) [fresh culture].
3. Incubate the fresh cultures in 37°C until the observed OD590 reaches 0.5.If the OD becomes over 0.5, dilute to 0.5 with LB medium.
4. Add the culture to LB medium as below.
　　LB medium contains 50 microg / mL ampicillin, 30 microg / mL kanamycin and 100 microg / mL chloramphenicol.
　　　• A 300 microL ＋ B 130 microL＋ LB medium 2.57 mL
　　　• A 300 microL ＋ LB medium 2.7 mL
　　　• B 130 microL ＋ LB medium 2.87 mL
5. Incubate these samples at 37°C for 6 h. (During that time, measure the optical density every one hour.)
6. Measure the fluorescence intensity with a flow cytometer (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company).

Co-culture Assay Result

Fig. 7. The growth of Customer(Plux-CmR-RhlI) and Campany(Prhl(RL)-CmR-RhlI) when co-cultured for 6 hours.

　Fig.7 shows the optical densities of Company and Customer after they were co-cultured for 6 hours.
These values were evaluated below:
　　The optical density of Company cell = the optical density after co-culturing for 6 h x the ratio of the cells with the fluorescence of GFP.
　　The optical density of Customer cell = the optical density after co-culturing for 6 h x the ratio of the cells without fluorescence of GFP.

　As a result, the co-cultured samples were able to grow better than the samples single cultured .
It means that only Customer cell can grow little by little in the medium with 100 microg/mL Chloramphenicol because of the leak of Plux promoter (Company cell can’t grow since it contains Prhl promoter which has less leak.)
However, in the co-culture experiment, Customer produced C4HSL by the leakage and it induced CmR and LasI expression of Company. LasI produced 3OC12HSL, and it induced CmR and RhlI expression of Customer.
This positive feedback might cause the mutualism of Company and Customer.

Fig. 8.Increase of OD(co-culture) / OD(mono culture)

To see how the initial amount affect the growth, an additional experiment was conducted.
We conducted the experiment shown above at a smaller scale.
Increase of OD(co-culture) / OD(mono culture) was calculated by the equation shown below.
　　　OD590 of the co-cultured sample / OD590 of the mono cultured sample
　　　　*OD was measured after 4h incubation
　　　　*The ratio of customer and company in co-cultured sample is Customer : Company = 30 : 13

Sample condition is as follows.

co-cultured sample	(53, 123)	(66, 154)	(83, 192)	(104, 240)
mono cultured sample	(53, 0) + (0,123)	(66, 0) + (0,154)	(83, 0) + (0,192)	(104, 0) + (0,240)

　　　　*(Initial cell concentration of Customer[microL], Initial cells concentration of Company[microL])

When the initial amount is enough, the increase of OD(co-culture) / OD(mono culture) is high because of the mutualism between Customer and Company.
On the contrary, the ratio is low when the initial amount is small (Fig. 8).

We operated three independent co-culture assays. First, we confirmed whether mutualism was achievable. This result shows that the final OD of co-cultured cells was more than the final OD of both mono-cultured cells combined, when the initial OD of Company (Prhl(RL)-CmR-LasI:BBa_K1529302) was 0.05 (Fig. 9).

Fig. 9.First Co-culture Assay

Secondly, we specified the range of the initial OD of Customer (Plux-CmR-RhlI:BBa_K1529797) that can achieve mutualism. This result shows that the best condition for mutualism is when the initial OD of Company (Prhl(RL)-CmR-LasI:BBa_K1529302) and Customer (Plux-CmR-RhlI:BBa_K1529797) were 0.05 and 0.02 (Fig. 10).

Fig. 10.Second Co-culture Assay

Thirdly, we confirmed whether we can achieve mutualism if the initial OD became lower. This result shows that the lower the initial OD was, the less possible the mutualism could be achieved (Fig. 11).

Fig. 11.Third Co-culture Assay

For more information, see [http://2014.igem.org/Team:Tokyo_Tech/Experiment/C4HSL-dependent_3OC12HSL_production our work in Tokyo_Tech 2014 wiki].

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