1. Summary

To accomplish our system, SMART E.coli, it is required that we make worker cells assemble to the guider cell. Then we tried to utilze a property known as chemotaxis. Chemotaxis lies on bacteria including Escherichia coli. They are believed to be attracted toward certain substances, including L-aspartate (L-Asp). We have tried to make an E. coli attracting other bacteria toward itself by a substrate-stimulated L-Asp production. When guider cells synthesize L-Asp, worker cells assembles around the guider cell and workers are able to toil effectively.Also, we characterized the chemoattraction of E.coli toward L-Asp, and compared the results to a computational simulation. We obtained supportive evidences for the agreement of the wet and the dry. Therefore, we propose that, if we get an E. coli secreting enough level of L-Asp, we can devise an inducible cell mustering system.

1.1. The production of L-Asp

AspA synthesizes L-Asp from fumaric acid and ammonia

In the past studies aiming at L-Asp over-production, the amount of L-Asp was determined by HPLC ^[1][2]. However, having no available HPLC-apparaturs, we were unable to use this method, so we tried to detect it in alternative ways.

We first tried to make an E. coli producing L-Asp using lac promoter BBa_R0011. WT transformed with BBa_K518004(lacP-RBS-AspA-d.Ter) was pre-cultured with 1mM IPTG. The culture was soaked in the fumaric acid solution containing annmonia. Note that AspA synthesizes L-Asp from fumaric acid and ammonia. The reaction mix was incubated at 37 degreed celcius for 1 hour. After the incubation, the concentration of L-Asp was measured through ninhydrin staining and ultraviolet-visible spectroscopy. Unfortunatelly, we could not gain obvious data. We had ninhydrin react with L-Asp produced by AspA. Ninhydrin probably reacted not only with L-Asp but also with remaining ammonia.

The second attempt was TLC. Two microlitters of the supernatant of the incubated reaction solution was spotted onto a TLC sillica plate, and extracted with 70% ethanol. We had expected L-Asp and ammonia to be separated. However, it was impracible to separate them using ethanol. We then tried various concentrations of acetone as a developing solvent, only to observe smearing lanes.

One of the main reasons of the failure is that our methods relied on ninhydrin reaction. Ninhydrin certaionly reacts with L-Asp. However, ninhydrin also reacts with an indispensable substance to AspA reaction. We should have selected a way that only one of reactants and products can be detected definitely. Now then, the sequencing result shows that Assembling BBa_K518004 was success. So, it may be possible to make sure of the work if a right method is selected. For example, L-Asp is detected by HTLC and AspA protain is detected instead of L-Asp.

1.2. The characterization of L-Asp chemotaxis

Next, to show that E. coli moves in the direction of higher L-Asp concentration, we carried out swarming assays. WT colony was innoculated into 0.25% agar LB plate and L-Asp solution was instilled. Plates were left at room temperture. After further 20 hours, those plates were captured. The representative photograph of a swarming colony is shown in Fig. 1A.

To determine the movement of colonies toward the location of L-Asp instillation, we then performed an image analsis. Obtained images were undergone a computational processing to find an edge of the colonies. The processed image is shown in Fig. 1B.

Fig. 1A: A swarming colony after 20-hour incubating. A cross "×" in the plate center was the position instilled L-Asp. Fig. 1B: A colony image processed to find the edge. Blue line is the border between a colony inner and outer.

In this assay, we defined a colony movement as a vector from the center of a colony to the intial tip position. The colony movements were presented in Fig. 1C. The migration length of colonies was measured about 38(±9) mm with L-Asp solution, while 13(±6) mm without L-Asp. The results clearly show that colonies exposed to the L-Asp solution swarmed significantly, compared to colonies of the control group. Data is obtained from more than 6 experiment.

Fig. 1C: movemant of colonies with L-Asp sol.(Asp+) and without L-Asp sol.(Asp-).

References

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