Team:ZJU-China/Project/ElvesCooperation/Results

Elves' Cooperation: Result

1.Characterize Chemotaxis of Detectors 2.Characterize Communication Between Detectors And Cleaners 3.Characterize the Function of Cleaners

1.Characterize Chemotaxis of Detectors The strain E.coli JW1870 (ΔcheZ) ,which was a generous gift from Prof. John S. Parkinson, University of Utah ,was mainly used for our experiments. The bacteria containing Plasmid 1 was incubated in LB medium with 100ug/mL Amp until mid-log phage. 10 uL suspension culture was diluted with 1mL LB medium and then dripped at the center of a semi-solid plate, which contained 0.2% agar , 100ug/mL Amp and was covered with 2mM atrazine on the surface. The plate was incubated at 37℃ for 10hrs. The negative control was required.

2.Characterize Communication Between Detectors And cleaners As what has been illustrated in Design part, Detector contains plasmid 1 and plasmid 2 while Cleaner contains plasmid 3.

Since GFP expression in Detector has been proved and detected in Characterization of Atrazine riboswitch. We directly verify the communication between Detectors and Cleaners by observing GFP and RFP with confocal microscope.

2.1LB media cultivation Both Detector and Cleaner were incubated in LB media to mid-log phage (OD600 reached 0.6 approximately) and then was induced by 0.2mM IPTG and 2mM atrazine simultaneously. Further incubation was conducted at 37℃ /180 rpm for 8hrs. The following images were photographed with confocal microscope after incubation. Negative control without Atrazine was still conducted. The RFP expression of Cleaner will be easily observed if it receives the signal molecule AHL, which is produced by Detector, successfully. However, our confocal images showed that RFP expression was not very strong and thus our proof of the elf’s communication was not that solid. Another thing illustrated in images was that GFP was also detected in negative control ,which indicated the existence of leakage.

2.2 Semisolid Medium Cultivation

An aliquot of suspension culture containing equivalent Detector and Cleaner was dripped to the center of semisolid plate (100ug/mL Amp and 0.2mM/L IPTG were added) whose surface was covered by 2mM Atrazine and then was incubated at 37℃ for 18hrs. Cells from the edge of colony were picked for confocal microscope observation. The confocal images revealed that neither GFP nor RFP was expressed substantially in either samples or negative control, which offered us no help to verify the communication between Detector and Cleaner.

Further experiments are still under conduction and we are full of confidence to find out the problem and justify our design.