Team:Chiba/Demo experiments:Receivers

Varying bacterial numbers: method

 * 1) Receiver(T9002) pre-incubation
 * 2) Receiver:BBa_T9002(JW1908)wascultured in 2mL LB-Amp (37&deg;C,12h)
 * 3) Pre-incubated Receiver(BBa_T9002(JW1908))was plated so as to produce about 1000 colonies.
 * 4) Sender(S03623) pre-incubation
 * 5) Sender:BBa_S03623(JW1908) was cultured in 50mL entrifuge tubes in 10mL of LB-Amp (37&deg;C,12h)(2 tubes)
 * 6) Sender Wash
 * 7) Centrifuged 2 tubes containing(BBa_T9002(JW1908))at 20&deg;C,3600rpm for 6min and discarded supernatant.
 * 8) Added 10mL LB-Amp to each tube.
 * 9) Repeated wash twice.
 * 10) Creating bacterial plates
 * 11) LB-Amp pre-cultured Sender(BBa_S03623(JW1908)) tube 1 (10mL) was mixed with LB-Amp-agar(50&deg;C)(10ml)to produce sender containing bacterialplate-1.
 * 12) LB-Amp pre-cultured Sender(BBa_S03623(JW1908)) tube 2(100&mu;l)was mixed with LB-Amp(9.9ml) and diluted 100-fold. 10ml of this solution was mixed with LB-Amp-agar(50&deg;C)(10ml) and created Sender(BBa_S03623(JW1908))containing bacterial plate-2.
 * 13) LB-Amp pre-cultured Sender solution-2(10&mu;l) and LB-Amp(9.99ml)was mixed to dilute 1000-fold.10ml of this solution and LB-Amp-agar(50&deg;C)(10ml) was mixed to create Sender(BBa_S03623(JW1908) containing bacterial plate-3
 * 14) Lifted with nitrocellulose
 * 15) Receiver(BBa_T9002(JW1908))colony was transfered to a nitrocellulose filter and placed on each of Sender(BBa_S03623(JW1908))containing bacterial plate (1~3) and Sender-absent negative control plate (t=0). Determined the time required for the colonies to fluoresce depending on the bacterial concentration (100 and 1000-fold dilution).
 * 16) Method to detect fluorescence
 * 17) Plates cultured at 37&deg;C were exposed to UV (312nm) light once every 30 minutes to observe GFP fluorescence.



Testing different receivers-methods

 * 1) Receiver&sender pre-culture
 * 2) Used Receivers were:
 * 3) *BBa_T9002:ptet-luxR-plux-GFP(high copy)
 * 4) *ptet-luxR-(low copy),BBa_J37032:plux-GFP(high copy)
 * 5) *BBa_T9002:ptet-luxR-plux-GFP(low copy)
 * 6) *ptet-mLuxR(too sensitive)-plux-GFP
 * 7) *ptet-luxR-plux-GFP-plac-aiiA
 * 8) *（all JW1908）Each was cultured in 2ml LB (37&deg;C,12h) and plated so that about 1000 colonies of receiver cells will grow.
 * 9) Sender:BBa_S03623(JW1908) was cultured in 10mL LB in 50mL centrifuge tubes (37&deg;C,12h)
 * 10) sender wash
 * 11) Each receiver-containing medium was centrifuged in 50mL tubes at de20&deg;C, 3600rpm for 6min and supernatant discarded.
 * 12) Added 10mL LB to each tube.
 * 13) Repeated wash twice.
 * 14) Creating bacterial plates
 * 15) LB pre-cultured Sender(BBa_S03623(JW1908)) tube 1 (10mL) was mixed with LB-agar(50&deg;C)(10ml)to produce sender containing bacterial plate-1.
 * 16) LB pre-cultured Sender(BBa_S03623(JW1908)) tube 2(100&mu;l)was mixed with LB(9.9ml) and diluted 100-fold. 10ml of this solution was mixed with LB-agar(50&deg;C)(10ml) and created Sender(BBa_S03623(JW1908)) containing bacterial plate-2.
 * 17) LB pre-cultured Sender solution-2(10&mu;l) and LB(9.99ml) was mixed to dilute 1000-fold.10ml of this solution and LB-agar(50&deg;C)(10ml) was mixed to create Sender(BBa_S03623(JW1908)) containing bacterial plate-3
 * 18) Lifted with nitrocellulose
 * 19) Each Receiver colony was transfered to a nitrocellulose filter and placed on a Sender(BBa_S03623(JW1908)) containing bacterial plate (1~3) or a sender-absent negative control plate(t=0) to observe how receiver type affects the time taken for the colonies to display visible fluorescence.
 * 20) Method to detect fluorescence
 * 21) Plates cultured at 37&deg;C were exposed to UV (312nm) light once every 30 minutes to observe GFP fluorescence.

results
No Dilution 0h                   　    0.5h                        1.0h

 100-fold dilution  0h                     0.5h　 　　　　　　　　　　　1.0h                       1.5h

 1000-fold dilution

0h                　  0.5h                1.0h　　　　　　　1.5h               2.0h

discussion

 * We demonstrated that the GFP expression switch is delayed by the ratio of sender to receiver.
 * The result indicates that the amount of AHL from one bacterium per time is constant and independent of bacteria number density.
 * This is probaly because the sender has no feedback circuit of AHL production.
 * Although this strategy can not change the time interval, we can manage the switch timing by changing the ratio of sender to receiver.

results
0h                0.5h                 1.0h                1.5h

1=N.C

2=BBa_T9002:ptet-luxR-plux-GFP(high copy)

3=ptet-luxR-(low copy),BBa_J37032 plux-GFP(high copy)

4=BBa_T9002:ptet-luxR-plux-GFP(low copy)

5=ptet-mLuxR(too sensitive)-plux-GFP

6=N.C

7=ptet-luxR-plux-GFP-plac-aiiA

discussion
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 * Number 2,3 and 5 fluoresces in 30 min. We could not see time difference of these, it may be resulted from excess amount of sender bacteria.
 * Precise experiments controlling the number of sender are necessary for further discussion.
 * In comparizon with number 2 (T9002:high copy), there was no change of fluorescence intensity of number 4 (T9002:low copy) in 4 hours.
 * It is probably because of circuit working, since the AHL is provided enough for the receiver.