1.Summary

Bacteria including Escherichia coli has a property known as chemotaxis. They are believed to be attracted toward certain substances, including L-aspartate (L-Asp). We have tryed to make an E. coli attracting other bacteria toward itself by a substrate-stimulated L-Asp production. 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

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 synthesize 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.

TBD

TBD