Difference between revisions of "Part:BBa K1529797:Experience"

Latest revision as of 19:21, 2 November 2014

Plux-CmR-RhlI

Materials and Methods

-Strain
All the samples were JM2.300 strain.

3OC12HSL-dependent CmR expression Protocol

1.Construction
A. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR-RhlI(pSB3K3)
B. Ptet-LuxR-Plac-RFP(pSB6A1), PlacIq-CmR (pSB3K3) (Positive control)

Fig. 1.Plasmids for the experiment of 3OC12HSL-dependent CmR expression.

2.Assay protocol
1.Prepare the overnight culture of cell A and B at 37°C.
2.Make a 1:100 dilution in 3 mL of fresh LB containing antibiotics and grow the cell 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 500 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 100microg / mL) + 30 microL C4HSL (final concentration is 500 microM)
　　　4) 3 mL of LB containing Amp, Kan and Cm (final concentration is 100microg / mL) + 30 microL DMSO
4. Grow the samples of sender cells at 37°C for more than 10 hours. Measure optical density every hour. (If optical density is over 1.0, dilute the cell medium.)

3OC12HSL-dependent C4HSL production Protocol

1.Construction
Sender
A. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR-RhlI(pSB3K3)
B. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR(pSB3K3)...Negative control
Reporter
C. Ptet-RhlR(pSB6A1), Plux-GFP(pSB3K3)
D. Ptet-RhlR(pSB6A1), PlacUV5-GFP(pSB3K3)...Positive control
E. Ptet-RhlR(pSB6A1), Promoter-less-GFP(pSB3K3)...Negative control

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

2.Assay protocol
Prepare the supernatant of the sender cell
1. Grow the colony of sender cell in LB containing antibiotic O/N at 37°C.
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 30 microL of 500 microM 3OC12HSL to 3 mL LB containing Amp and Kan
　　　b) Add 30 microL DMSO to 3 mL LB containing Amp and Kan
4 .Grow the samples of sender cell at 37°C for 8 hours.
5. Centrifuge sample at 9000x g, 4°C for 1minute. Filter sterilize supernatant. (Pore size is 0.22 microm. ) Use this supernatant in reporter assay.

Reporter Assay
1. Grow the colony of Reporter cell (described upper) in LB containing antibiotic (Amp and Kan) over night at 37°C.
2. Make a 1:100 dilution in 3 mL of fresh LB+ antibiotics and grow the cells at 37°C until you reach an 0.5 in OD590 (fresh culture).
3. Add 30 microL of the culture containing reporter cell 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 Ba +300 microL LB
　　　4) 2.7 mL filtrate of Bb +300 microL LB
　　　5) 3 mL LB + 500 microM C4HSL 30 microM (final concentration is 5 microM)
　　　6) 3 mL LB + DMSO 30 microL
4. Grow the samples of Reporter cell in incubator at 37°C for 4 hours.
5. Start preparing the flow cytometer 1 h before the end of incubation.
6. After 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

3OC12HSL-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 and Fig. 4 show the condition in the absence and the presence of chloramphenicol, respectively.

• 

  Fig. 3. 3OC12HSL-dependent CmR expressionh in no chloramphenico

• 

  Fig. 4. 3OC12HSL-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 Plux-CmR-RhlI can grow only when it was induced by 3OC12HSL.
Without the induction of 3OC12HSL, the cell cannot express CmR and cannot grow in the presence of chloramphenicol.
As a result, we confirmed that Plux-CmR-RhlI expressed CmR when induced by 3OC12HSL as expected.

3OC12HSL-dependent C4HSL production Result

　Fig. 5 shows the fluorescence intensities generated by reporter cells.
When the reporter cell C (Plux-CmR-RhlI) was incubated in the condition (1) (the culture of the induced Customer 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 95-fold higher fluorescence intensity.

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

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

Co-culture Assay

　From the results so far, Customer cell containing Plux-CmR-RhlI expresses CmR and RhlI in the presence of 3OC12HSL.
This enables Customer to survive and to produce C4HSL. (This result is showed above.)
On the other hand, Company cell containing Prhl(RL)-CmR-LasI(BBa_K1529302) expresses CmR and LasI in the presence of C4HSL.
This enables Company to survive and to produce 3OC12HSL. (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.

　　　　*(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. 6).

Fig. 6.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. 7).

Fig. 7.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. 8).

Fig. 8.Third Co-culture Assay

For more information, see [http://2014.igem.org/Team:Tokyo_Tech/Experiment/3OC12HSL-dependent_C4HSL_production our work in Tokyo_Tech 2014 wiki].

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