Team:Chiba/Demo experiments:Senders



Method

 * 1) Pre-culture
 * 2) Picked and cultured the following glycerol stocks in 2mL of LB:
 * 3) LB-Amp, BBa_T9002, (JW1908)
 * 4) LB-Amp+0.2%Glu, BBa_K084012(plac+rbs+LuxI(no LVA)), (XL10G)
 * 5) LB-Amp+0.2%Glu, BBa_K084007(plac+rbs+LasI(no LVA)), (XL10G)
 * 6) Cultured at 37&deg;C for 12h.
 * 7) Culture
 * 8) Added 6.25% each of the pre-cultures to new LB medium.
 * 9) LB-Amp, BBa_T9002
 * 10) LB-Amp+0.2%Glu, BBa_K084012(plac+rbs+LuxI(no LVA)), BBa_K084007(plac+rbs+LasI(no LVA))
 * 11) Cultured at 37&deg;C for 4~5h
 * 12) Wash
 * 13) Transfer 10mL each of the culture to 50mL centrifuge tubes.
 * 14) Centrifuged for 6min at 3600rpm,20&deg;C and discarded the supernatant.
 * 15) Added LB-Amp to each centrifuge tube:
 * 16) 10mL to the tube that contains BBa_T9002
 * 17) 5mL to the tube that contains BBa_K084012, BBa_K084007
 * 18) Centrifuged for 6min, 3600rpm at 20&deg;C the tube containing BBa_K084012, BBa_K084007 and discarded　the supernatant.
 * 19) 10mL to the tube that contains BBa_K084012, BBa_K084007
 * 20) Centrifuged for 6min, 3600rpm at 20&deg;C the tube containing BBa_K084012, BBa_K084007 and discarded　the supernatant.
 * 21) 5mL to the tube that contains BBa_K084012, BBa_K084007
 * Mix
 * 1) Mixed the sender cells BBa_K084012 and BBa_K084007 both with BBa_T9002 at a 1:1 ratio.
 * 2) Added 100&mu;L each to a 96-well shallow plate (as shown in the figure).
 * 3) Green part is BBa_K084012:BBa_T9002=1:1
 * 4) Red part is BBa_K084007:BBa_T9002=1:1
 * 5) Uncolored part is BBa_T9002 alone.
 * 6) Culture and observe results

Results
Green region: sender=LuxI, Red circular region: sender=Las I.

LuxI GFP is detected at 4h following mixing while LasI GFP is detected after 8h, thus successfully demonstrating time-delay depending on the sender used.