Team:ZJU-China/Notebook/TheElf/Results

The "Elf": Results

Characterize Chemotaxis of Detectors

The strain E.coli R01616 (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.

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.

LB 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.

A B C D E F

(A) GFP and RFP were both expressed when atrazine was added into LB medium. (B) GFP was expressed when detector recognized atrazine. (C) Communication between detector and cleaner was characterized by RFP expression. (D/E) Negative control was performed without IPTG and atrazine, GFP was weakly observed which indicated the existence of leakage. (F) RFP was too weak to be observed.

L denotes for LB sample, Ln represents for LB negative control

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.

A B C D E F

(A) Both IPTG and atrazine were added in to semisolid, the expression of GFP was strong while RFP was weakly. (B) GFP was expressed when detector recognized atrazine. (C) Communication between detector and cleaner was unsatisfactory. (D/E) Negative control was performed without IPTG and atrazine, GFP was weakly observed which indicated the existence of leakage. (F) RFP was too weak to be observed.

"S"means semisolid sample,"sn"means semisolid negative control. Further experiments are still under conduction and we are full of confidence to find out the problem and justify our design.