Team:Chiba/Calendar-Home/23 October 2008

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22 October 2008 <|> 24 October 2008

Varying bacterial numbers

 * 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

 * 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.