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Our Project

Modeling

Modeling by UT-Tokyo
We consigned modeling of our project to UT-Tokyo.

fig.1

This model means random walk of E.coli on swarming plate.The vertical axis means arbitrary unit.The horizontal axis means spatial axis.

Method

Medium

Aspartate synthesis

E.coli K12 transformed with CdP-R.B.S-AspA-d.Ter (BBa_K1342001) previous cultured with cadmium in LB medium (250μM) in 37℃ for 12hr at 120rpm. Adjust Cell mass (OD1.0) and therefor centrifuged 4000rpm for 20 min. Cell pellets ware activated in synthesis medium in 37℃ for 2hr at 120rpm/min.

SDS PAGE

・Preculture E.coli holding a plasmid containing a target gene or nomal E.coli.
・Measure OD600 0.6-1.0
・the expression of fusion protein by isopropylthio-β-D-galactoside (IPTG)　and Cd2+ soln.
・Transfer a sample a 200 µl in a microcentrifuge tube
・Centrifuge at 13,000rpm for a minute at 4℃
・Discard supernatant quantitative
・Store pellet at -20 °C
・Thaw pellet and resupend in Sample Buffer (100 µL 1xSample Buffer per samples)
・Heat for 5 minutes at 98 °C
・Centrifuge at 13,000rpm for 10 minutes at 4℃
・Transfer supernatant to a new microcentrifuge tube
・Analyze samples by SDS-PAGE.(Use 20 µL per samples)

TLC assay

We analyzed L-aspartate by thin-layer chromatography (TLC). The same amount added Supernatant of synthesis medium to 7mg/mL 5-Dimethylamino naphthalene-1-sulfonyl Chloride (in Acetone) and incubated more than 30min. Two microliters of the sample was placed onto a TLC silica plate, and developed in a mixture of Ethyl acetate, pyridine, water, and acetic acid(162：21：11：6 V/V).

Chemotaxis Assay

Swarmming Assay

Capillary Assay

Chemotaxis of a bacterium such as E.coli is assayed by measuring the number of organisms attracted into a capillary tube containing an attractant.
Preculture JM109 using tripton broth (Incubate 50rpm/min 30℃ 12hr) Check motility under the microscope.Diluting the E.coli culture 100 times. (tripton broth:E.coli culture＝20mL:200㎕)Incubate 50rpm/min 30℃ (until log phase)500㎕ into two tubes.Centrifuge two tubes in low speed. (25℃ 10min in 3400G)Throw away the clear top of liquid.Add wash medium(500㎕)Resuspending pellets softlyDo 5~9 againAdd chemotaxis medium(500㎕)Resuspending pellets softly.Check motility under the microscope.E.coli chemotaxis medium into chamber preparation(100㎕)Prepare the positive and negative control capillary.（1㎕ of 10mM aspartate chemotaxis medium and chemotaxis medium into capillary.）Two capillary into chamber preparation.90mim 30℃ incubationDilute the chemotaxis buffer in capillary`s liquid 100 times.Prating to LBmedium.37℃ 12h incubation.Counting the colony.Finish

Result & Discussion

TLC Assay

Analysis of aspartate from strain E.coli K-12/BBa_K1342001 by thin-layer chromatography (TLC) Lane: 1, not addition cadmium to previous culture of synthesis medium (diluted 1:50 by synthesis medium) 2, (diluted 1:100 by synthesis medium) 3, add cadmium (250μM) 4, (diluted 1:50 by synthesis medium) 5, (diluted 1:100 by synthesis medium) 6, (diluted 1:200 by synthesis medium)

SDS PAGE

1. marker 2. 0hr 3. 0.5hr 4. 2hr 5. 6hr 6. 24hr 7. Marker marker:Precision Plus Protein Standards (BIO-RAD)

OD600=0.74 CdCl2 100μM/IPTG 1mM

Future work

Reference