From 2014.igem.org
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| - | ==='Method'===
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| - | <br>
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| - | '''Aspartate synthesis'''
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| - | 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.
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| - | TLC
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| - | <br>
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| - | ・Developer
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| - | <br>
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| - | Ethyl acetate: pyridine: water: acetic acid＝162：21：11：6
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| - | <br>
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| - | Reagent
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| - | 7mg/mL 5-(Dimethylamino) naphthalene-1-sulfonyl Chloride (in Acetone); (DNS)
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| - | <br>
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| - | 1.Sample: DNS =1: 1
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| - | <br>
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| - | 2.Incubate RT ＞30min
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| - | 3.Spot 2μL
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| - | <br>
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| - | 4.Development
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| - | 5.UV irradiation (365nm)
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| - | <br>
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| - | '''SDS PAGE'''
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| - | <br>
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| - | ・Preculture E.coli holding a plasmid containing a target gene or nomal E.coli.
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| - | <br>
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| - | ・Measure OD600 0.6-1.0
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| - | ・the expression of fusion protein by isopropylthio-β-D-galactoside (IPTG) and Cd2+ soln.
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| - | ・Transfer a sample a 200 µl in a microcentrifuge tube
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| - | ・Centrifuge at 13,000rpm for a minute at 4℃
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| - | ・Discard supernatant quantitative
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| - | <br>
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| - | ・Store pellet at -20 °C
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| - | <br>
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| - | ・Thaw pellet and resupend in Sample Buffer (100 µL 1xSample Buffer per samples)
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| - | <br>
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| - | ・Heat for 5 minutes at 98 °C
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| - | <br>
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| - | ・Centrifuge at 13,000rpm for 10 minutes at 4℃
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| - | ・Transfer supernatant to a new microcentrifuge tube
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| - | <br>
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| - | ・Analyze samples by SDS-PAGE.(Use 20 µL per samples)
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| - | <font size=5>Chemotaxis Assay Using Capillary</font>
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| - | <font size=3>Chemotaxis of a bacterium such as E.coli is assayed by measuring the number of organisms attracted into a capillary tube containing an attractant.</font>
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| - | <font size=4>Protocol</font>
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| - | <font size=3>
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| - | 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
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| - | </font>
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Revision as of 09:17, 15 October 2014
BBa_K1342001
BBa_K1342002
BBa_K1342003
BBa_K1342004
BBa_K1342005
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