Team:Nagahama project

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Abstract

One E.coli Has One Function

What is our project at 2014?

Cadmium is one of harmful materialｓ for us. 50 years ago, itai-iati disease was going around in center of Japan Gihu. The cause was industrial wastewater. Cadmium contained the water. Our project is to find a way to clean contaminated with Cadmium. we think how to clean the water. We use E.coli. Concretely, we use two kinds of E.coli. One catches Cadmium.　the Other makes to all E.coli to use chemoattractant. Catches E.coli displays metallothionein (metallothionein is a protein that combines a heavy metal. Cadomium is one kind of heavy metal). Other is releasing Asp (Asp is one kind of chemoattractant. All E.coli is close to the Asp E.coli). To use these E.coli. Finally cadomiun is catched(wastewater is be clean).

What experiment we did?

・How to check E.coli's chemotaxis? We make use of other igem team’s　assay. Using soft agar plate.we let E.coli siwm.

・We made a plasmid Our project need to two kind of plasmid. we made one plasmid.And we have to make one more plasmid.

Method

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.

TLC
・Developer
Ethyl acetate: pyridine: water: acetic acid＝162：21：11：6
Reagent

7mg/mL 5-(Dimethylamino) naphthalene-1-sulfonyl Chloride (in Acetone); (DNS)
1.Sample: DNS =1: 1
2.Incubate RT ＞30min
3.Spot 2μL
4.Development
5.UV irradiation (365nm)
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)

Chemotaxis Assay Using Capillary

Chemotaxis of a bacterium such as E.coli is assayed by measuring the number of organisms attracted into a capillary tube containing an attractant.

Protocol

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

fig1

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 CdCl　100μM/IPTG 1mM