Team:Nagahama project

From 2014.igem.org

(Difference between revisions)
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=== Method ===
=== Method ===
 +
Aspartate synthesis
 +
#2mL LB medium + CdCl2 final concentration 250μM
 +
#Overnight 37℃ shaking
 +
#Adjust Cell mass (OD1.0)
 +
#4000rpm 20min
 +
#1.5mL Synthesis medium + cell pellet
 +
#Incubate in 37℃
TLC
TLC

Revision as of 07:12, 13 October 2014


Abst


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

  1. 2mL LB medium + CdCl2 final concentration 250μM
  2. Overnight 37℃ shaking
  3. Adjust Cell mass (OD1.0)
  4. 4000rpm 20min
  5. 1.5mL Synthesis medium + cell pellet
  6. Incubate in 37℃

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)

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